©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Ubiquitin C-terminal Hydrolases from Chick Skeletal Muscle (*)

(Received for publication, January 17, 1995; and in revised form, June 2, 1995)

Seung Kyoon Woo (1) Jae Il Lee (1) Il Kyoo Park (1) Yung Joon Yoo (2) Choong Myung Cho (2) Man-Sik Kang (1) Doo Bong Ha (1) Keiji Tanaka (3) Chin Ha Chung (1)(§)

From the (1)Department of Molecular Biology, SRC for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea, (2)Lucky Biotech Ltd., Taejon 305, Korea, and the (3)Institute for Enzyme Research, Tokushima University, Tokushima 770, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A new method for assaying ubiquitin C-terminal hydrolases was developed using a I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a nondenaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and from ubiquitin-alphaNH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin(RGA)-alphaNH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.


INTRODUCTION

Ubiquitin is a highly conserved and the most widely distributed eukaryotic polypeptide(1) . This 76-amino acid protein is ligated covalently to a variety of intracellular proteins by a family of ubiquitin-conjugating enzymes, called E2s(2, 3) . An isopeptide linkage is formed between the C-terminal Gly residue of ubiquitin and the -amino group of the Lys residue(s) of proteins. Ubiquitins by themselves or which have already been conjugated to proteins may also be ligated to additional ubiquitin molecules to form branched polyubiquitin by the linkage between the -amino group of Lys-48 of one ubiquitin and the C terminus of the other. This ubiquitination has been implicated in the regulation of a variety of cellular processes such as selective protein breakdown(4, 5) , cell cycle regulation(6, 7, 8, 9) , and stress response(10, 11, 12) . In addition, the dynamic nature of the ubiquitin-protein conjugate pool has been demonstrated in vivo by microinjection (13, 14) and immunochemical techniques(15, 16) . These studies have shown that the reversible ubiquitination of proteins is under the control of external stimuli such as heat shock and starvation. Therefore, the enzymes that remove ubiquitins proteolytically from ubiquitin-protein conjugates should be of importance in maintaining the steady-state levels of free ubiquitin for a variety of its cellular functions.

In all eukaryotic cells, ubiquitins are encoded by two distinct classes of gene, neither of which encodes the monomeric form of ubiquitin (17, 18, 19) . One is a polyubiquitin gene that encodes a polyprotein of up to 100 uninterrupted, in tandem repeated ubiquitins through peptide bonds between the C-terminal Gly and N-terminal Met of contiguous ubiquitin molecules. The other encodes a fusion protein of which a single ubiquitin is linked to a ribosomal protein consisting of 52 or 76-80 amino acids. The transient association of ubiquitin with the ribosomal proteins has been suggested to promote their incorporation into ribosomes(20) . Therefore, proteolysis at the peptide bonds between ubiquitin and carboxyl extension proteins is required to generate ribosomal proteins for ribosome biogenesis as well as free ubiquitins.

A number of the genes encoding ubiquitin C-terminal hydrolases (UCHs) (^1)have been cloned from Saccharomyces cerevisiae. Miller et al.(21) cloned the gene for a YUH1 protease in S. cerevisiae which releases ubiquitin from its linear C-terminal conjugates to relatively short peptides. Varshavsky and co-workers (22, 23) cloned three different genes for yeast ubiquitin-specific proteases named UBP1, UBP2, and UBP3, which also hydrolyze linear ubiquitin conjugates irrespective of the size of their C-terminal polypeptides. Papa and Hochstrasser (24) demonstrated that the yeast DOA4 (UBP4) gene and the human tre-2 oncogene encode deubiquitinating enzymes, both of which can release ubiquitin molecules that are conjugated to proteins by alphaNH-peptide bonds or NH-isopeptide linkages. These studies with yeast containing at least five different deubiquitinating enzymes imply the existence of a variety of uncharacterized UCHs also in higher eukaryotic organisms.

A family of UCHs, named L1, L2, L3, and H2, has been identified from bovine calf thymus using a small ubiquitin C-terminal adduct (i.e. ubiquitin-O-C(2)H(5)) as a substrate(25) . Of these, L3 has been shown to be a mammalian homolog of the yeast YUH1 protease(26) . However, only two of the enzymes (L1 and L3) have been purified so far, partly because of the difficulty and/or insensitivity of the available assay methods. Therefore, a simple and sensitive assay method is of necessity for facilitating the purification of UCHs, for studying more systematically the diversity of the enzymes in a single source, and for comparison of the properties of the enzymes from different sources.

Rechsteiner and co-workers have constructed ubiquitin-alphaNH-peptide extensions containing ``PEST'' sequences(27, 28) . In the present study, for assaying UCHs, we used one of the ubiquitin extensions, of which the peptide portion (MHISPPEPESEEEEEHYC) can be almost exclusively radioiodinated and is short enough to be released as acid-soluble products upon hydrolysis of the ubiquitin-peptide by UCHs. Using this assay protocol we show that the extracts of chick skeletal muscle contain at least 10 UCHs that appear to be distinct from each other in their chromatographic behavior, size, and substrate specificity, and we purify one of them to apparent homogeneity.


EXPERIMENTAL PROCEDURES

Materials

Heparin-Sepharose, phenyl-Superose, and Superose-6 and -12 were obtained from Pharmacia Biotech Inc.; NaI from DuPont NEN; and IODO-BEADS from Pierce. pQE31 vector for poly-His tagging and Ni-nitrilotriacetic acid-agarose were obtained from Qiagen. All other agents were purchased from Sigma unless otherwise indicated. pUbq1 containing the rice hexaubiquitin gene (29) was obtained from Dr. Y.-M. Kim (Institute for Agricultural Development, Korea).

Protein Purification

YUH1 was purified from Escherichia coli cells carrying pYUH1 as described(21) . pYUH1 was prepared using two oligonucleotide primers, derived from the published sequence of YUH1(21) . Ubiquitin-alphaNH-MHISPPEPESEEEEEHYC (henceforth referred to as Ub-PESTc) was purified from E. coli strain AR13 carrying pNMHUB-PESTc as described by Yoo et al.(27) . Ubiquitin(RGA)-alphaNH-PARRKWQKTGHAVRAIGRLSS (Ub(A)-P-MLCK20) was purified from E. coli strain M15 carrying pUbMLCK as described(27) . In this ubiquitin extension peptide, the C-terminal Gly of ubiquitin was replaced by Ala, and a Pro residue was inserted into the N terminus of the extension peptide, which is the 20-amino acid sequence of the calmodulin binding domain of myosin light chain kinase. Ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids (Ub-CEP80) was purified from E. coli strain AR58 carrying pNMHUB-CEP80 as described by Monia et al.(30) . Ubiquitin-alphaNH-dihydrofolate reductase (Ub-DHFR) was purified from E. coli strain M15 carrying pDSUb-DHFR using a methotrexate affinity matrix as described by Tobias and Varshavsky(22) . Poly-His-tagged diubiquitin was purified from E. coli strain M15 carrying pQEDiUB using Ni-nitrilotriacetic acid resin by following the standard procedure supplied by the manufacturer. pQEDiUB was prepared by ligating the XhoI restriction fragment of pUbq1 into pQE31 after gap filling with Klenow. The 20 and 26 S proteosomes were purified from chick skeletal muscle as described previously(31, 32) .

Radioiodination of Ub-PESTc

Purified Ub-PESTc was radiolabeled with NaI using IODO-BEADS(33) . Briefly, 0.5 mg of purified Ub-PESTc was incubated for 30 min at room temperature in 0.2 ml of 0.1 M sodium phosphate buffer (pH 7) containing two IODO-BEADS and 0.5 mCi of NaI. After incubation, the radiolabeled Ub-PESTc was separated from free iodine by gel filtration on a Sephadex G-25 column equilibrated with the phosphate buffer. Ub(A)-P-MLCK20 was radiolabeled with NaI using chloramine T(34) .

Assay for Hydrolysis of Ub-PESTc

Reaction mixtures (0.1 ml) contained proper amounts of purified YUH1 or the chromatographic fractions of muscle extract (see below) and 1 µg of I-labeled Ub-PESTc (10^4 cpm/µg) in 100 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol (DTT), and 5% (v/v) glycerol. After incubating the mixtures for various periods at 37 °C, the reaction was terminated by adding 50 µl of 40% (w/v) trichloroacetic acid and 50 µl of 1.2% (w/v) bovine serum albumin. The samples were centrifuged, and the resulting supernatants were counted for their radioactivity using a gamma-counter. The enzyme activity was then expressed as a percentage of I-labeled Ub-PESTc hydrolyzed to acid-soluble products.

Preparation of Muscle Extract

Chick pectoralis muscle tissues (150 g) were minced and homogenized using a Waring blender in buffer A (25 mM Tris-HCl buffer (pH 7.8) containing 1 mM DTT, 1 mM EDTA, and 10% glycerol). The homogenates were centrifuged at 10,000 g for 1 h to remove cell debris, and their supernatants were centrifuged again at 100,000 g for 2 h. The resulting supernatants were titrated with 1 M Tris base to pH 7.8 and referred to as the muscle extract.

Preparation of Ubiquitin-NH-Protein Conjugates

Extracts of Xenopus oocytes were prepared by washing the eggs twice with 10 mM Hepes (pH 7.7) containing 50 mM sucrose, 0.1 mM CaCl(2), 100 mM KCl, 1 mM MgCl(2), and 0.1 mg/ml cytochalasin B and then by centrifugation at 12,000 g for 20 min(35) . The supernatant fraction was added with an ATP-regenerating system consisting of 15 units/ml creatine phosphokinase, 6.6 mM phosphocreatine, 0.5 mM ATP, 10 mM Tris-HCl (pH 7.8), 0.5 mM MgCl(2), 1 mM KCl, and 0.05 mM DTT and kept frozen at -70 °C.

To prepare ubiquitin-NH-protein conjugates, Ub(A)-P-MLCK20 was radioiodinated using chloramine T(34) . The oocyte extracts (0.6 mg) were incubated with 0.4 µg of the I-labeled Ub(A)-P-MLCK20, 10 µg of free ubiquitin, and the ATP-regenerating system for 2 h at 30 °C and then for 10 min at 55 °C. Precipitates were removed by centrifugation, and the heat-stable supernatant fraction containing ubiquitin-NH-conjugates of Ub(A)-P-MLCK20 was used as a substrate for UCHs.

Electrophoresis

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol was performed as described by Laemmli (36) or using Tris-Tricine buffer as described by Schägger and von Jagow(37) . The discontinuous slab gels contained 4, 10, and 16% (w/v) polyacrylamide to improve resolution of small proteins. The sample buffer contained 150 mM Tris-HCl (pH 6.8), 1.5% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 0.002% (w/v) bromphenol blue, and 7% glycerol. After electrophoresis, the gels were stained with Coomassie Blue R-250 or covered with Saran Wrap and exposed directly to x-ray films (Fuji) at -70 °C.

Amino Acid Sequence Analysis

Partially purified UCHs from muscle extracts (see below) were incubated with I-labeled Ub-PESTc, and aliquots (0.2 ml) of the incubation mixtures were subjected to gel filtration on a Sephadex G-50 column (1 40 cm) equilibrated with buffer A. Fractions of 1 ml were collected at a flow rate of 8 ml/h and counted for their radioactivity. Fractions with high radioactivity were pooled, concentrated by ultrafiltration using a YM2 membrane (Amicon), and subjected to Edman degradation for determination of their N-terminal amino acid sequences using ProSequencer (model 6600, MilliGen).


RESULTS

Assay of UCHs UsingI-Labeled Ub-PESTc

To develop a simple and rapid method for assaying UCHs, we first examined whether purified YUH1 can convert I-labeled Ub-PESTc into acid-soluble products. Fig.1shows that the production of radioactive, acid-soluble materials increases in an incubation time-dependent manner, and this cleavage reaction can be almost completely blocked by preincubation with iodoacetamide, an inhibitor of YUH1(21) . Since YUH1 is known to cleave specifically the carboxyl side of the C-terminal RGG sequence of ubiquitin, the acid-soluble products should represent the PESTc peptides released from Ub-PESTc.


Figure 1: Time-dependent hydrolysis of I-labeled Ub-PESTc by purified YUH1. Reaction mixtures contained 0.3 µg of YUH1 and 1 µg of I-labeled Ub-PESTc (1-2 10^4 cpm/µg) in 100 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM DTT, and 5% glycerol. The mixtures were incubated for various periods at 37 °C in the absence () and presence of 1 mM iodoacetamide (IAA, bullet). After incubation, the radioactivity released as acid-soluble products was determined as described under ``Experimental Procedures.''



The 26 S protease complex has been reported to have deubiquitinating activity in the presence of ATP(38) . Therefore, we examined whether purified 26 S and/or 20 S proteosomes can also convert I-labeled Ub-PESTc into acid-soluble product. Since the casein-degrading activity of the 20 S proteosome is known to be activated by poly-L-Lys or fatty acids(39, 40) , the assays were performed in the presence and absence of poly-L-Lys. Little or no hydrolysis of the substrate was observed with either the 26 or 20 S proteosome whether or not ATP was supplied (Table1). However, we fortuitously found that poly-L-Lys could markedly increase the Ub-PESTc-hydrolyzing activities of YUH1 and the extracts of chick skeletal muscle. Therefore, the assays for separation of the UCH activities in the muscle extract were performed in the presence and absence of poly-L-Lys.



Separation of UCH Activities in Muscle Extract

From 150 g of chick pectoralis muscle, approximately 4.2 g of protein of the tissue extract was obtained. The extract was applied to a DEAE-Sepharose column (2.5 20 cm) equilibrated with buffer A containing 0.1 M NaCl. After collecting the flow-through fractions, proteins bound to the column were eluted with a linear gradient of 0.1-0.3 M NaCl. Every third of the column fractions was assayed for its ability to cleave I-labeled Ub-PESTc. Two broad peaks of UCH activities were eluted, and both were stimulated markedly by the poly-L-Lys treatment (Fig.2). Fractions under peak I which did not bind to the DEAE column in the presence of 0.1 M NaCl were pooled and added with solid ammonium sulfate to a final concentration of 55%. Precipitated proteins were recovered by centrifugation and dialyzed against buffer A. The recovery of UCH activities from peak I by this ammonium sulfate step was approximately 90% (data not shown), and therefore no further studies were made for the proteins soluble in the presence of 55% ammonium sulfate.


Figure 2: Separation of UCH activities in the extract of chick skeletal muscle using a DEAE-Sepharose column. The muscle extract (4.2 g of protein) was chromatographed on a DEAE-Sepharose column (2.5 20 cm) as described under ``Results.'' Fractions of 20 ml were collected at a flow rate of 150 ml/h, and aliquots (20 µl each) of them were assayed for their ability to hydrolyze I-labeled Ub-PESTc in the absence () and presence of 0.1 mg/ml poly-L-Lys (bullet). Incubations were performed for 30 min at 37 °C. The slashed line shows the NaCl gradient, and the dotted line indicates the protein profile.



The dialyzed proteins were loaded again onto the same DEAE-Sepharose column but equilibrated with buffer A (i.e. without NaCl). After collecting the flow-through fractions, proteins bound to the column were eluted with 0.25 M NaCl (Fig.3A). The UCH activity seen in the flow-through fractions was tentatively named UCH-1 since it remained as a single peak upon further chromatographic trials on other columns (see below). The fractions under the bar in Fig.3A were pooled, dialyzed against buffer A, and loaded onto a heparin-Sepharose column (2.5 16 cm) equilibrated with the same buffer. After collecting the flow-through fractions, proteins bound to the column were eluted with 0.4 M NaCl (Fig.3B). The heparin flow-through fractions were pooled, added with NaCl to a final concentration of 0.15 M, and loaded onto a Mono Q column (0.5 5 cm) equilibrated with buffer A containing 0.15 M NaCl (Fig.3C). Proteins bound to the column were then eluted with a linear gradient of 0.15-0.35 M NaCl. Two peaks of UCH activity were eluted and named UCH-2 and UCH-3 in the order of their elution. The heparin-bound fractions in Fig.3B were pooled, dialyzed against buffer A containing 50 mM NaCl, and applied to a HiLoad-Q column (2.6 10 cm) equilibrated with the same buffer. Proteins bound to the column were then eluted with a linear gradient of 0.05-0.4 M NaCl (Fig.3D). A symmetric peak of activity (UCH-4) was eluted at a NaCl concentration of 0.12 M, in addition to multiple peaks of UCH activity eluting at higher salt concentrations (UCH-5s). Because of their unstability no further attempts were made to separation the latter activity peaks.


Figure 3: Separation of UCH activities in peak I obtained from the first DEAE-Sepharose column. The active fractions under peak I from the first DEAE-column (see Fig.2) were pooled and added with solid ammonium sulfate to 55% saturation. The precipitated proteins were dialyzed against buffer A and separated further by chromatography on DEAE-Sepharose (panel A), heparin-Sepharose (panel B), Mono Q (panel C), and HiLoad-Q columns (panel D) as described under ``Results.'' Hydrolysis of I-labeled Ub-PESTc was then assayed by incubating appropriate amounts of the column fractions for 0.5-2 h at 37 °C in the absence () and presence of 0.1 mg/ml poly-L-Lys (bullet). The slashed lines show the NaCl gradients, and the dotted lines indicate the protein profiles. The numbers on the top of each activity peak represent the individuals or mixtures of the UCHs in peak I from the first DEAE-Sepharose column.



Fractions under peak II from the first DEAE column (see Fig.2) were pooled, dialyzed against buffer A, and chromatographed on a heparin-Sepharose column (1 10 cm) equilibrated with the same buffer. After collecting the flow-through fractions, proteins bound to the column were eluted with a linear gradient of 0-0.6 M NaCl. At least three peaks of UCH activity were eluted from the column (Fig.4A): the first one in the flow-though fractions (UCH-6), the broad second peak eluting from about 30 to 350 mM NaCl, and the third eluting at about 0.5 M NaCl (UCH-10). The fractions under the bar (i.e. the broad second peak) were pooled, concentrated by ultrafiltration, and subjected to chromatography on a Superose-6 column (1 30 cm) equilibrated with buffer A. As shown in Fig.4B, the sample was again separated into three distinct peaks (UCH-7, UCH-8, and UCH-9 in the order of their elution). Except UCH-5s, all other UCH activities could not be separated further by other columns so far tested, including hydroxylapatite, phosphocellulose, and gel filtration columns (data not shown). Therefore, these activities appear to represent distinct UCHs in chick skeletal muscle at least in their chromatographic behavior.


Figure 4: Separation of UCH activities in peak II obtained from the first DEAE-Sepharose column. The active fractions under peak II from the first DEAE-column (see Fig.2) were pooled, dialyzed against buffer A, and subjected to chromatography on heparin-Sepharose (panel A) and Superose-6 columns (panel B) as described under ``Results.'' Hydrolysis of Ub-PESTc was then assayed by incubating appropriate amounts of the column fractions for 0.5-1 h at 37 °C in the absence () and presence of 0.1 mg/ml poly-L-Lys (bullet). The slashed line shows the NaCl gradient, and the dotted lines indicate the protein profile. The numbers at the top of each activity peak represent the individuals or mixtures of UCHs in peak II from the first DEAE-Sepharose column.



Purification of UCH-6

When each of the partially purified UCHs was pooled and compared for its total activity against Ub-PESTc, the activity of UCH-6 constituted at least 50% of the total activity of the 10 enzymes (data not shown). Therefore, we proceeded to purify the major, Ub-PESTc-degrading activity in muscle extract. The pooled fractions of UCH-6 from the heparin-Sepharose column (see Fig.4A) were dialyzed against 25 mM NaH(2)PO(4)/Na(2)HPO(4) buffer (pH 7.5) containing 1 mM DTT, 1 mM EDTA, and 10% glycerol and loaded on a hydroxylapatite column (1.5 9 cm) equilibrated with the same buffer. Proteins that did not bind to the column were pooled, dialyzed against buffer A containing 1 M ammonium sulfate, and loaded on a phenyl-Superose column (0.5 5 cm) equilibrated with buffer A containing 1 M ammonium sulfate. After washing the column, proteins bound to the column were then eluted with a reverse gradient from 1 M to 1 mM ammonium sulfate (Fig.5A). Fractions with high activity were pooled, concentrated by ultrafiltration using YM10 membrane (Amicon), and dialyzed against buffer A containing 50 mM NaCl. The sample was then loaded on a Superose-12 column (1 30 cm) equilibrated with the dialysis buffer. The fractions under the symmetric peak of UCH-6 activity (Fig.5B) were pooled, concentrated by ultrafiltration, and kept frozen at -70 °C until use. Upon analysis of the enzyme sample by polyacrylamide gel electrophoresis in the presence of SDS under reducing condition, only a single band could be seen in the gel. This result indicates that UCH-6 was purified to apparent homogeneity. The overall purification protocols are summarized in Table2.


Figure 5: Separation of UCH-6 using phenyl-Superose and Superose-12 columns. The UCH-6 preparation obtained from the heparin-Sepharose column (see Fig.4A) was subjected to three successive chromatographies on hydroxylapatite, phenyl-Superose (panel A) and Superose-12 columns (panel B) as described under ``Results.'' Hydrolysis of Ub-PESTc was then assayed by incubating appropriate amounts of the column fractions for 30 min at 37 °C in the absence () and presence of 0.1 mg/ml poly-L-Lys (bullet). The size markers used are: a, alcohol dehydrogenase (150 kDa); b, bovine serum albumin (66 kDa); c, carbonic anhydrase (29 kDa); d, cytochrome c (12.4 kDa). The slashed line shows the ammonium sulfate gradient, and the dotted lines indicate the protein profile. Active fractions from the Superose-12 column were pooled, and an aliquot (7 µg) of the pooled sample was subjected to electrophoresis on a 12% polyacrylamide slab gel containing SDS and 2-mercaptoethanol followed by staining with Coomassie Blue R-250 (panel C).





Size Estimation of Muscle UCHs

Upon gel filtration analysis of purified UCH-6 on the Superose-12 column (see Fig.5B), the native size of the enzyme was estimated to be 54 kDa. However, when the same enzyme preparation was subjected to polyacrylamide gel electrophoresis under denaturing conditions, it behaved as a 27-kDa polypeptide (Fig.5C). Thus, UCH-6 appears to consist of two identical subunits of 27 kDa.

To determine the sizes of the other partially purified muscle UCHs under nondenaturing conditions, each of them was subjected to gel filtration on either a Superose-6 or Superose-12 column (1 30 cm) that had been equilibrated with buffer A containing 50 mM NaCl. Fractions of 0.5 ml were collected and assayed for their ability to cleave I-labeled Ub-PESTc in the presence of 0.1 mg/ml poly-L-Lys. Apparent sizes of the UCHs were then estimated by running standard size markers on the same gel filtration columns, and they are as follows: UCH-1, 35 kDa; UCH-2, 50 kDa; UCH-3, 45 kDa; UCH-4, 100 kDa; UCHs 6, 9, and 10, 54 kDa; UCH-7, 810 kDa; UCH-8, 200 kDa. However, the subunit sizes of the muscle UCHs, except UCH-6, remain to be determined until after each of them was purified to near homogeneity.

Effect of Poly-L-Lys on Purified UCH-6 Activity

To determine more precisely the effect of poly-L-Lys on the activity of purified UCH-6 against I-labeled Ub-PESTc, we first measured the incubation time-dependent hydrolysis of the substrate in the presence and absence of 0.1 mg/ml poly-L-Lys. Fig.6A shows that the hydrolysis of Ub-PESTc increases linearly with time at least for 40 min. Therefore, the effects of increasing concentrations of poly-L-Lys were then determined by incubating purified enzyme for 30 min with the same amount of substrate (Fig.6B). From these data, the concentration of poly-L-Lys which gives a half-maximal stimulatory effect was estimated to be 2.2 µg/ml.


Figure 6: Effect of poly-L-Lys on the hydrolysis of I-labeled Ub-PESTc by purified UCH-6. Left panel, in the absence () and presence of 0.1 mg/ml poly-L-Lys (bullet), the activity of purified UCH-6 (20 ng) was assayed by incubation with 1 µg of Ub-PESTc at 37 °C for various periods. Right panel, UCH-6 activity was assayed as above but by incubating the enzyme for 30 min in the presence of increasing amounts of poly-L-Lys.



Since the peptide portion of Ub-PESTc contains 7 Glu and 2 His residues and therefore should be highly negative at the assay condition, it appeared possible that the stimulatory effect of poly-L-Lys is due simply to its ability to neutralize the negative charge. To test this possibility, the enzyme assays were performed in the presence of a variety of polyionic agents. As shown in Table3, poly-L-Arg stimulated the hydrolysis of I-labeled Ub-PESTc by UCH-6 nearly as well as poly-L-Lys. However, other polycations, including histone, spermine, and putrescine, showed little or no effect on enzyme activity. Moreover, aninoic poly-L-Glu showed no effect. These results suggest that the stimulatory effect of poly-L-Lys on the Ub-PESTc-degrading activity of UCH-6 and the other UCHs in muscle is not due to simple charge effect. Purified UCH-6 was maximally active between pH 7 and 7.5 and nearly inactive at pH below 6 and above 9.



Cleavage Specificity of Muscle UCHs

To determine whether partially purified muscle UCHs can generate free ubiquitin and PESTc peptide, each of the enzyme preparations was incubated with I-labeled Ub-PESTc and poly-L-Lys for 2 h at 37 °C and then heated to 85 °C for 10 min to precipitate off the proteins in the incubation mixtures from the heat-stable substrate and its cleavage products. The supernatant fractions were then subjected to electrophoresis on discontinuous polyacrylamide slab gels containing SDS followed by staining with Coomassie Blue R-250. As shown in Fig.7A, all of the partially purified UCHs as well as YUH1 were capable of generating free ubiquitin from Ub-PESTc. However, no PESTc peptide band could be detected, perhaps due to diffusion of the small peptide out from the gels during the Coomassie staining procedure. Therefore, the same incubation mixtures treated as above were again electrophoresed, and the resulting gels were exposed directly to x-ray films. Fig.7B clearly shows that the PESTc peptides are also produced by partially purified muscle UCHs as well as YUH1. Nearly identical data were obtained when the same experiments were performed with purified UCH-6 (data not shown). However, the PESTc peptide generated by UCH-2 and UCH-5s appeared to be degraded further by certain unidentified protease(s) and/or peptidase(s) that may be contaminated in the partially purified enzyme preparations.


Figure 7: Hydrolysis of I-labeled Ub-PESTc by partially purified UCHs from muscle extract and electrophoretic analysis of their products. Aliquots (2-10 µl depending on their specific activities) of partially purified UCHs were incubated with 5 µg of Ub-PESTc and 0.1 mg/ml poly-L-Lys for 2 h at 37 °C. After incubation, the mixtures were heated for 10 min at 85 °C and centrifuged for 10 min at 12,000 g to remove precipitates. The supernatants were then electrophoresed in duplicate on discontinuous slab gels containing SDS and 2-mercaptoethanol as described under ``Experimental Procedures.'' One of the gels was stained with Coomassie Blue R-250 (panel A), and the other gel was exposed directly to x-ray film for autoradiography (panel B). Lane a indicates 5 µg of unlabeled free ubiquitin; lane b, I-labeled Ub-PESTc incubated alone; lane y, incubated with 0.4 µg of purified YUH1; lanes 1-10, incubated with partially purified UCHs from muscle extract. The arrowhead indicates the cleavage product of the PESTc peptide.



To determine whether the muscle UCHs indeed specifically cleave the alphaNH-peptide bond between ubiquitin and PESTc, the incubation mixtures were prepared as above and loaded onto a Sephadex G-50 column equilibrated with the assay buffer. Fractions of 1 ml were collected and counted for their radioactivity. As shown in Fig.8, a new peak of radioactivity which corresponds to a size of about 2 kDa was generated by UCH-1 and UCH-6 with a concomitant reduction in the radioactivity peak of I-labeled Ub-PESTc. The fractions with high radioactivity under the second peak were pooled and subjected to Edman degradation. The N-terminal 9-amino acid sequence of the peptide products was MHISPPEPE, which is identical to that of the PESTc peptide. We performed the same experiments for all other UCHs except UCH-2 and UCH-5s and obtained identical results (data not shown). These results clearly indicate that the UCHs from chick skeletal muscle specifically cleave the alphaNH-peptide bond between ubiquitin and the PESTc peptide.


Figure 8: Separation of the PESTc peptides generated by partially purified UCH-1 and UCH-6 and determination of their N-terminal amino acid sequences. I-Labeled Ub-PESTc (20 µg) was incubated for 3 h at 37 °C without () and with 360 µg of partially purified UCH-1 (up triangle, filled) or 8 µg of UCH-6 (bullet). After incubation, the samples were loaded on a Sephadex G-50 column (1 40 cm) equilibrated with buffer A. Fractions of 1 ml were collected and counted for their radioactivity. The new radioactivity peaks generated by incubation with the UCHs were pooled and subjected to Edman degradation for determination of their N-terminal amino acid sequence as described under ``Experimental Procedures.'' The resulting 9-amino acid sequence is shown at the top of the radioactivity peaks.



Substrate Specificity of Muscle UCHs

To determine whether purified UCH-6 can generate free ubiquitin from other ubiquitin-alphaNH-extension proteins, the enzyme was incubated with Ub-CEP80 and Ub-DHFR at 37 °C for 2 h. We also incubated purified YUH1 with the substrates to compare its activity with that of UCH-6. After incubation, the samples were subjected to electrophoresis on discontinuous slab gels containing SDS followed by staining with Coomassie Blue R-250. Fig.9shows that UCH-6 is capable of hydrolyzing both of the substrates, whereas YUH1 cleaves a negligible amount of Ub-DHFR in accord with the earlier report(23) . In addition, YUH1, even in the presence of twice the amount of UCH-6, hydrolyzed Ub-CEP80 approximately 30% as well as the latter enzyme as determined by scanning the ubiquitin bands in the gel using a densitometer.


Figure 9: Hydrolysis of Ub-CEP80 and Ub-DHFR by UCH-6 and YUH1. Purified UCH-6 (0.5 µg; lanes c) and YUH1 (1 µg; lanes d) were incubated with 5 µg of Ub-CEP80 (panel A) or Ub-DHFR (panel B) at 37 °C for 2 h. After incubation, the samples were subjected to electrophoresis on discontinuous slab gels as described in the legend of Fig.7. The proteins in the gels were stained with Coomassie Blue R-250. Free ubiquitin (lanes a), Ub-CEP80 (lane b in panel A), and Ub-DHFR (lane b in panel B), which were incubated by themselves, are also shown. The dots indicate the position where the purified enzymes migrated. The Ub-DHFR preparation is contaminated by an unknown protein (arrowhead) having exactly the same size as DHFR (22 kDa). Note that the intensity of the 22-kDa band increases upon hydrolysis of Ub-DHFR by UCH-6.



We also examined whether partially purified muscle UCHs can generate free ubiquitin from other ubiquitin-alphaNH-extension proteins. Each of the enzyme preparations was incubated with Ub-CEP80 and Ub-DHFR as above, heated at 85 °C for 10 min, and centrifuged at 12,000 g for 10 min. The heat-stable supernatant fractions were then subjected to electrophoretic analysis as above. All of the muscle UCHs were capable of generating ubiquitin molecules from both Ub-CEP80 and Ub-DHFR (data not shown). Without poly-L-Lys, Ub-CEP80 was hydrolyzed at approximately the same rates as Ub-PESTc by the muscle UCHs, except UCH-6, by which Ub-PESTc was cleaved about 10-fold more rapidly than Ub-CEP80. In addition, at least 2-5-fold higher amounts of the enzymes were required for generation of the same amounts of free ubiquitin from Ub-DHFR as that from Ub-PESTc. When similar experiments were performed with His-tagged diubiquitin, only five of the enzyme preparations, UCHs 1 to 5s, could release free ubiquitin from the substrate (data not shown).

To determine whether any of the UCHs can hydrolyze the isopeptide linkage, partially purified UCHs were incubated with the ubiquitin-conjugates of I-labeled Ub(A)-P-MLCK20, in which the C-terminal Gly residue of ubiquitin was replaced by Ala, and a Pro residue was inserted to the N terminus of MLCK20 for preventing the action of the enzyme preparations on the alphaNH-peptide bond between ubiquitin and the extension peptide. As shown in Fig.10, only UCH-1 and UCH-7 were capable of removing ubiquitin from monoubiquitinated Ub(A)-P-MLCK20. However, little hydrolysis of di- or triubiquitinated Ub(A)-P-MLCK20 was observed by the enzyme preparations or by other muscle UCHs. These results suggest that UCH-1 and UCH-7 may cleave off only the ubiquitin molecules that are linked directly to proteins by isopeptide bonds. Unlike the hydrolysis of Ub-PESTc by UCHs 2 to 10, however, poly-L-Lys showed little or no stimulatory effect on the cleavage of any of Ub-CEP80, Ub-DHFR, His-tagged diubiquitin, or the monoubiquitinated Ub(A)-P-MLCK20 (data not shown). Thus, it remains unclear how poly-L-Lys can specifically activate the Ub-PESTc-degrading activities of YUH1 and the muscle UCHs, except UCH-1.


Figure 10: Cleavage of isopeptide linkages in ubiquitinated Ub(A)-P-MLCK20 by partially purified muscle UCHs. I-Labeled Ub(A)-P-MLCK20 was ubiquitinated on its NH-Lys group as described under ``Experimental Procedures.'' Muscle UCHs were incubated with the ubiquitinated Ub(A)-P-MLCK20 at 37 °C for 4 h. After incubation, the samples were subjected to electrophoresis on discontinuous gels as described in Fig.7. The resulting gels were then exposed to x-ray films for autoradiography. The arrowheads from top to bottom indicate tri-, di-, and monoubiquitinated Ub(A)-P-MLCK20.



Effects of Protease Inhibitors and KCl on UCH Activities

We then examined the effect of various site-specific protease inhibitors and KCl on the activities of the UCHs against I-labeled Ub-PESTc (Table4). Similar to the other known UCHs(25) , all of the muscle UCHs are also highly sensitive to inhibition by sulfhydryl blocking reagents such as iodoacetamide. In addition, the activities of all of the UCHs, except UCH-1, were strongly inhibited by high salt concentrations such as 0.3 M KCl or NaCl, although they could be recovered fully upon dialysis to remove the salt. On the other hand, little or no inhibition was observed by treatment of serine protease inhibitors, including diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. In addition, ATP showed no effect on the activity of any of the UCHs (data not shown). The effects of the inhibitors and KCl on partially purified UCH-6 were identical to those seen with purified enzyme. Similar results were obtained when the sensitivity of the UCHs to the protease inhibitors was assayed using Ub-CEP80, Ub-DHFR, or His-tagged diubiquitin as the substrate (data not shown). These results suggest that the 10 newly identified UCHs appear different from each other in both physical and biochemical criteria such as their sizes and inhibitor sensitivity, in addition to their distinct chromatographic behavior. However, it is still possible that some of the UCHs may be derived from partial degradation of a given UCH(s) since chick muscle is known to contain significant amounts of proteases.




DISCUSSION

In the present studies, a simple method was developed for assaying UCHs using I-labeled Ub-PESTc as a substrate. When the substrate was incubated with purified YUH1 or chick muscle extract, the I-labeled PESTc peptide portion was readily released into acid-soluble products. Of interest was the finding that most of the radioactivity in the substrate could be recovered from the acid-soluble fraction upon incubation for prolonged periods or with increasing amounts of the enzymes (see Fig.1and Table 1). These results indicate that radioiodination of Ub-PESTc under a mild condition, such as using IODO-BEADS, occurs almost exclusively to the PESTc peptide portion (most likely at the 17th Tyr residue), despite the fact that ubiquitin itself also contains a Tyr residue. This finding allows us to assay UCH activities rapidly and to quantify precisely the cleavage products. Furthermore, the assay protocol was validated by electrophoretic analysis of the cleavage products and by N-terminal sequencing of the PESTc peptide. Therefore, by simple measurement of radioactivity of the PESTc peptide released into the acid-soluble fraction we were able to identify at least 10 chromatographically distinct UCHs in chick muscle extracts and to purify one (UCH-6) of them to apparent homogeneity.

Of particular interest was the observation that poly-L-Lys markedly stimulated the Ub-PESTc-cleaving activities of purified YUH1 and the newly identified muscle UCHs, except UCH-1. Furthermore, treatment of other short polycationic agents, including spermine, putrescine, and histones, showed little or no effect on the activity of purified UCH-6 and the other partially purified enzymes, suggesting that the stimulatory effect of poly-L-Lys is not due to a simple charge interaction between the poly-L-Lys and the PESTc peptide. The 20 S proteosome, which is known to be a proteolytic core of the ATP/ubiquitin-dependent 26 S proteosome, has been suggested to be a latent enzyme since it degrades proteins such as casein only in the presence of poly-L-Lys or fatty acids(39, 40) . In this regard, we initially thought that the muscle UCHs might also be latent enzymes that could be activated under appropriate conditions. However, poly-L-Lys showed little or no stimulatory effect on the activity of any of the 10 muscle UCHs against Ub-CEP80, Ub-DHFR, His-tagged diubiquitin, or monoubiquitinated Ub(A)-P-MLCK20. Therefore, the mechanism by which poly-L-Lys stimulates the Ub-PESTc-cleaving activities of the muscle UCHs (except UCH-1) as well as of purified YUH1 remains unclear.

All of the UCHs identified in the present studies using I-labeled Ub-PESTc as a substrate were also capable of releasing free ubiquitin from Ub-CEP80 and Ub-DHFR. Moreover, in the absence of poly-L-Lys, Ub-CEP80 was hydrolyzed by the individual UCH, except UCH-6, more or less at the same rate as Ub-PESTc. Thus, muscle UCHs may play a role in the generation of both ribosomal proteins and free ubiquitins. On the other hand, Ub-DHFR was hydrolyzed by all of the muscle UCHs much less efficiently than Ub-PESTc or Ub-CEP80. In addition, UCH-6 cleaved Ub-PESTc much faster than Ub-CEP80. These findings suggest that the size and/or the tertiary structure of extension proteins may affect the susceptibility of ubiquitin-alphaNH-protein extensions to the muscle enzymes. In contrast to the ubiquitin extension proteins, His-tagged diubiquitin was cleaved by only five of the enzymes (UCHs 1 to 5). Therefore, the five UCHs may also participate in the generation of free ubiquitin molecules from the product of the polyubiquitin gene in muscle cells, although it has been suggested that the processing of polyubiquitin may occur cotranslationally in yeast(23) .

In addition to the alphaNH-peptide bond cleaving activity, UCH-1 and UCH-7 were also capable of hydrolyzing the isopeptide linkage of monoubiquitinated Ub(A)-P-MLCK20. These enzymes or other muscle UCHs, however, showed little or no activity against di- or triubiquitinated Ub(A)-P-MLCK20. Hershko and co-workers (41) have shown that the isopeptidase T removes ubiquitins from high molecular weight, multiubiquitinated protein conjugates but not from low molecular weight forms. They also have demonstrated that the 26 S proteosome has inherent deubiquitinating activity against adducts of which a single ubiquitin is linked to NH-Lys group of protein as well as against conjugates containing multiple ubiquitins(38) . Therefore, UCH-1 and UCH-7 together with the 26 S proteosome may be involved in the release of ubiquitin from the Lys residue of the end products generated by the action of the isopeptidase T against the polyubiquitinated protein conjugates for complete recycling of ubiquitin molecules.

However, determination of the activities against ubiquitinated Ub(A)-P-MLCK20 as well as against Ub-CEP80 and Ub-DHFR was done with partially purified muscle UCHs, which were separated from each other using Ub-PESTc as the substrate. Therefore, we could not exclude the possibility that these activities are due to other contaminated enzymes active against the substrates other than Ub-PESTc. Much effort on complete purification of the muscle UCHs and isolation of their cDNAs is required for detailed characterization of the enzymes, for clarification of identity of the enzymes with the previously reported UCHs from other sources, and ultimately for their functional analysis.


FOOTNOTES

*
This work was supported by grants from the Korea Science and Engineering Foundation through SRC for Cell Differentiation and the Ministries of Education of Korea and Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 82-2-880-6693; Fax: 82-2-872-1993.

^1
The abbreviations used are: UCH, ubiquitin C-terminal hydrolase; YUH1, yeast ubiquitin hydrolase 1; Ub-PESTc, ubiquitin-alphaNH-MHISPPEPESEEEEEHYC; DTT, dithiothreitol; MLCK, myosin light chain kinase; CEP80, carboxyl extension protein 80; DHFR, dihydrofolate reductase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Rechsteiner (University of Utah School of Medicine) for providing E. coli strain AR13 carrying pNMHUB-PESTc, to Dr. Y.-M. Kim for pUbq1 containing the rice hexaubiquitin gene (Institute for Agricultural Development, Korea), and to Dr. Young Mok Park (Center for Supporting Basic Science, Korea) for N-terminal sequence analysis of the PESTc peptide. We also thank Dong Hun Shin and Sung Hee Baek for help in certain experiments.


REFERENCES

  1. Rechsteiner, M. (1987) Annu. Rev. Cell Biol. 3,1-30 [CrossRef]
  2. Pickart, C. M. (1988) in Ubiquitin (Rechsteiner, M., ed) pp. 77-100, Plenum Press, New York
  3. Jentsch, S., Seufert, W., Sommer, T., and Reins, H.-A. (1990) Trends Biochem. Sci. 15,195-198 [CrossRef][Medline] [Order article via Infotrieve]
  4. Hershko, A. (1991) Trends Biochem. Sci. 16,265-268 [CrossRef][Medline] [Order article via Infotrieve]
  5. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem. 61,761-807 [CrossRef][Medline] [Order article via Infotrieve]
  6. Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37,43-55 [Medline] [Order article via Infotrieve]
  7. Hochstrasser, M., Ellison, M. J., Chau, V., and Varshavsky, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4606-4610 [Abstract]
  8. Jentsch, S., McGrath, J. P., and Varshavsky, A. (1987) Nature 329,131-134 [CrossRef][Medline] [Order article via Infotrieve]
  9. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Nature 349,132-138 [CrossRef][Medline] [Order article via Infotrieve]
  10. Bond, U., and Schlesinger, M. J. (1986) Mol. Cell. Biol. 6,4602-4610 [Medline] [Order article via Infotrieve]
  11. Finley, D., Ozkaynak, E., and Varshavsky, A. (1987) Cell 48,1035-1046 [Medline] [Order article via Infotrieve]
  12. Joslin, G., Hafeez, W., and Perlmutter, D. H. (1991) J. Immunol. 147,1614-1620 [Abstract/Free Full Text]
  13. Carlson, N., and Rechsteiner, M. (1987) J. Cell Biol. 104,537-546 [Abstract]
  14. Carlson, N., Rogers, S., and Rechsteiner, M. (1987) J. Cell Biol. 104,547-555 [Abstract]
  15. Haas, A. L., and Bright, P. M. (1985) J. Biol. Chem. 260,12464-12473 [Abstract/Free Full Text]
  16. Haas, A. L. (1988) in Ubiquitin (Rechsteiner, M., ed) pp. 173-206, Plenum Press, New York
  17. Ozkaynak, E., Finley, D., and Varshavsky, A. (1984) Nature 312,663-666 [Medline] [Order article via Infotrieve]
  18. Lund, P. K., Moats-Staats, B. M., Simmons, J. G., Hoyt, E., D'Ercole, A. J., Martin, F., and Van Wyk, J. J. (1985) J. Biol. Chem. 260,7609-7613 [Abstract/Free Full Text]
  19. Ozkaynak, E., Finley, D., Solomon, M. J., and Varshavsky, A. (1987) EMBO J. 6,1429-1439 [Abstract]
  20. Finley, D., Bartel, B., and Varshavsky, A. (1989) Nature 338,394-401 [CrossRef][Medline] [Order article via Infotrieve]
  21. Miller, H. I., Henzel, W. J., Ridgway, J. B., Kuang, W.-J., Chisholm, V., and Liu, C.-C. (1989) Bio/Technology 7,698-704
  22. Tobias, J. W., and Varshavsky, A. (1991) J. Biol. Chem. 266,12021-12028 [Abstract/Free Full Text]
  23. Baker, R. T., Tobias, J. W., and Varshavsky, A. (1992) J. Biol. Chem. 267,23364-23375 [Abstract/Free Full Text]
  24. Papa, F. R., and Hochstrasser, M. (1993) Nature 366,313-319 [CrossRef][Medline] [Order article via Infotrieve]
  25. Mayer, A. N., and Wilkinson, K. D. (1989) Biochemistry 28,166-172 [Medline] [Order article via Infotrieve]
  26. Wilkinson, K. D., Lee, K., Deshpande, S., Duerksen-Hughes, P., Boss, J. M., and Pohl, J. (1989) Science 246,670-673 [Medline] [Order article via Infotrieve]
  27. Yoo, Y., Rote, K., and Rechsteiner, M. (1989) J. Biol. Chem. 264,17078-17083 [Abstract/Free Full Text]
  28. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234,364-368 [Medline] [Order article via Infotrieve]
  29. Kim, Y.-M., Kim, J.-K., and Hwang, Y.-S. (1994) Plant Physiol. 106,791-792 [Free Full Text]
  30. Monia, B. P., Ecker, D. J., Jonnalagadda, S., March, J., Gotlib, L., Butt, T. R., and Crooke, S. T. (1989) J. Biol. Chem. 264,4093-4103 [Abstract/Free Full Text]
  31. Seol, J. H., Park, S. C., Ha, D. B., Chung, C. H., Tanaka, K., and Ichihara, A. (1989) FEBS Lett. 247,197-200 [CrossRef][Medline] [Order article via Infotrieve]
  32. Lee, D. H., Kim, S. S., Kim, K. I., Ahn, J. Y., Shim, K. S., Nishigai, M., Ikai, A., Tammura, T., Tanaka, K., Ichihara, A., Ha, D. B., and Chung, C. H. (1993) Biochem. Mol. Biol. Int. 30,121-130 [Medline] [Order article via Infotrieve]
  33. Markwell, M. A. K. (1982) Anal. Biochem. 125,427-432 [Medline] [Order article via Infotrieve]
  34. Greenwood, F. C., Hunter, W. H., and Glover, J. (1963) Biochem. J. 89,114-123
  35. Murray, A. W., and Kirschner, M. W. (1989) Nature 339,275-280 [CrossRef][Medline] [Order article via Infotrieve]
  36. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  37. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166,368-379 [Medline] [Order article via Infotrieve]
  38. Eytan, E., Armon, T., Heller, H., Beck, S., and Hershko, A. (1993) J. Biol. Chem. 268,4668-4674 [Abstract/Free Full Text]
  39. Dahlmann, B., Ruschmann, M., Kuehn, L., and Reinauer, H. (1985) Biochem. J. 228,171-177 [Medline] [Order article via Infotrieve]
  40. Tanaka, K., Ii, K., Ichihara, A., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261,15197-15203 [Abstract/Free Full Text]
  41. Hadari, T., Warms, J. V. B., Rose, I. A., and Hershko, A. (1992) J. Biol. Chem. 267,719-727 [Abstract/Free Full Text]

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