©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Requirements for Transglutaminases
INFLUENCE OF THE AMINO ACID RESIDUE PRECEDING THE AMINE DONOR LYSINE IN A NATIVE PROTEIN (*)

(Received for publication, May 5, 1995; and in revised form, July 11, 1995)

Johan J. Grootjans Patricia J. T. A. Groenen Wilfried W. de Jong (§)

From the Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Thirteen recombinant alphaA-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacity of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes. It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alphaA-crystallin for transglutaminases. This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect. This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases. It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins.


INTRODUCTION

Transglutaminases (TGs) (^1)form a ubiquitous family of structurally and functionally related enzymes (for recent overviews see (1, 2, 3, 4) ). In man and other mammals, five enzymatically active TGs have been identified: the catalytic a subunit of plasma factor XIII, keratinocyte TG (TGK or TGase1), tissue TG (TGC or TGase2), epidermal TG (TGE or TGase3), and prostate TG (TGP or TGase4). These enzymes catalyze the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and can attach polyamines to peptide-bound glutamines. Their most obvious function is to stabilize biological structures. There is increasing evidence, however, that especially TGC is involved in many additional, more subtle biological processes, like apoptotic cell death (5) and tumor progression(6) . Intriguing recent examples of unexpected functional roles are the involvement of TGs in activation of phospholipase A(2)(7) , in the regeneration of optic nerves(8) , and as a GTP-binding protein in alpha(1)-adrenergic receptor signaling(9) .

The transglutaminase cross-linking reaction involves a Ca-dependent acyl transfer that follows a double displacement mechanism(10, 11) . In the first step, a glutamine-containing acyl donor substrate binds to the enzyme. Nucleophilic attack of the active site thiol of the enzyme on the -carboxamide group of a glutamine residue in the substrate protein leads to formation of an acyl-enzyme intermediate, under release of ammonia. The amine donor substrate then binds, and the acyl group is transferred to the primary amine, resulting in release of the -glutamyl amine product. Elucidation of the three-dimensional structure of factor XIII identified a catalytic Cys-His-Asp triad and suggests how the glutamine and lysine substrate proteins can approach each other in the active site cavity(12) . However, direct information about the substrate binding regions of the TGs is still lacking.

The number of proteins acting as glutaminyl substrates for TGs is restricted. Both primary structure and conformation of a protein appear to determine whether a glutamine residue can be reactive(10, 13, 14) . A considerable number of reactive glutamine residues have been identified in various substrate proteins for TGs(14, 15) . Accessibility of these glutamines in solvent-exposed surface regions or flexible extensions of the protein is a logical common feature. There is no obvious consensus sequence around these substrate glutamines, although some regularities can be discerned.

A common notion is that TGs are much less selective toward amine donor lysine residues in proteins than they are to the glutamine substrates (1, 3) . It appears indeed, on basis of studies with various primary amines, that TGC and factor XIII have a broad tolerance to structural differences in amine donor substrates(10, 11) . However, the modes of interaction of TGs with small amines and with native protein substrates are likely to be different(10, 16) . The limited number of identified amine donor lysine residues in substrate proteins for TGs(17, 18) precludes as yet an assessment of the substrate requirements in native proteins.

To fill this gap we apply site-directed mutagenesis to study the primary structure requirements around amine donor lysines in proteins, using alphaA-crystallin as a model. This demonstrated already that TGC accepts a variety of residues flanking the substrate lysine in alphaA-crystallin, albeit to different extents(17) . The present work focuses on a more detailed analysis of the influence of the amino acid side chain present directly NH(2)-terminal of the substrate lysine. On the basis of studies with synthetic peptides it has been proposed that this side chain might be a major determinant in acyl enzyme-lysine substrate interaction(10, 19, 20) . We wanted, moreover, to compare the reactivity of TGC and factor XIII with the various mutant substrate proteins, because these enzymes have been reported to have markedly different specificities(10) . Because first substrates may predicate amine specificity(10) , we also assessed the influence of using different acyl donor substrates.


MATERIALS AND METHODS

Preparation of Mutant alphaA-Crystallin cDNA Constructs

The strategy for mutagenesis was as described previously(17) . As starting material the expression clone pET8calphaA containing the full-length cDNA of bovine alphaA-crystallin (21) was used. Mutant alphaA-crystallin constructs were made using degenerated HhaI-BamHI linkers encoding the COOH-terminal amino acid residues (positions 171-174) to be mutated (see Fig. 1). The XbaI-HhaI insert of pET8calphaA, coding for amino acid residues 1-170, and a degenerated HhaI-BamHI linker were simultaneously cloned into XbaI/BamHI-digested pGEM7Zf(+) plasmid (Promega). Mutant clones were selected by sequencing these constructs, using the Sequenase version 2.0 DNA sequencing kit (U. S. Biochemicals). Identified mutant clones were plated and again sequenced. From these double sequenced clones, we isolated the XbaI-BamHI inserts, which encode the entire mutant alphaA-crystallins, and cloned them in the XbaI/BamHI-digested expression vector pET8c. This provided the pET8c constructs to express the mutant alphaA-crystallins. The Escherichia coli strains JM 109 and DH5alpha were used as recipients for the plasmid constructs in the cloning experiments.


Figure 1: Degenerated linkers used to construct the mutant alphaA-crystallins. The wild-type (wt) HhaI-BamHI fragment from clone pET8calphaA encodes the three carboxyl-terminal amino acid residues of bovine alphaA-crystallin (positions 171-173) and contains the translation termination codon. Substituting this wild-type HhaI-BamHI fragment with the degenerated HhaI-BamHI linkers 1-4 generated expression clones for the mutant alphaA-crystallins, introducing an amine donor lysine at position 173 and a variable residue in front of it.



Isolation of Mutant alphaA-Crystallins

Expression of wild-type and mutant alphaA-crystallins in the host E. coli BL21(DE3) and preparation of the water-soluble fraction were performed essentially as described(21) . Wild-type and mutant alphaA-crystallins were separated from the E. coli proteins by fast flow DEAE-Sepharose column chromatography (Pharmacia). The water-soluble fraction was dialyzed against the starting buffer (20 mM Tris, 20 mM NaCl, pH 7.5) and loaded onto a 25-ml column. Elution at 0.5 ml/min was performed with 50 ml of the starting buffer, followed by 100 ml of 20 mM Tris buffer forming a linear gradient of 100-300 mM NaCl, pH 7.5. Fractions of 5 ml were collected. The wild-type and mutant alphaA-crystallins eluted between 80 and 90 ml. 1 ml of the top fraction was taken to perform the transglutaminase assay. The remainder of the peak fractions was dialyzed against water and lyophilized. Between 10 and 20 mg of protein was obtained from a 250-ml culture.

Determination of Amine Donor Substrate Capacities for Transglutaminase

A hexapeptide (TVQQEL) and a heptapeptide (PGGQQIV) were biotinylated (22) and used as amine acceptor probes in the transglutaminase assay. For the TG reaction, 20 µl of the DEAE-Sepharose top fraction (containing approximately 20 µg of protein) was brought to a final volume of 50 µl in 0.05 M Tris, pH 7.5, 0.1 M NaCl, 12 mM CaCl(2), 2 mM leupeptine (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Merck) containing 2 mM amine acceptor probe. In the case of TGC, the mixture was made 20 volume % in glycerol. The reactions were started by adding 5 milliunits of guinea pig liver transglutaminase (Sigma) or 0.25 E coagulation factor XIII (Behring Werke) activated with 1 milliunit of thrombin from human plasma (Sigma). Incubations were performed at 37 °C and stopped after 3 h by adding EDTA to a final concentration of 24 mM.

To quantitate the reactivity of the proteins toward the amine acceptor probes, two samples (10 µl) of each incubation were analyzed in parallel by SDS-polyacrylamide gel electrophoresis. One gel was used for staining with Coomassie Brilliant Blue (CBB) and the other for streptavidin blot analysis(22) . These pairs of gels were prepared in triplicate. All CBB-stained gels and streptavidin blots were scanned twice with an LKB-Bromma laser densitometer. Measurements were in the linear range of densities. For each sample the average and standard deviation for the CBB staining and the streptavidin reaction were calculated. The streptavidin values for each mutant were normalized using the corresponding CBB densities to correct for gel loading variations and differences in protein concentrations between the samples.


RESULTS AND DISCUSSION

The aim of the present work was to study the influence on TG reactivity of the type of amino acid side chain present directly NH(2)-terminal of the substrate lysine in a native protein. To that end we constructed 13 mutant clones of bovine alphaA-crystallin, encoding for proteins having the COOH-terminal sequence Ala-Leu-Xaa-Lys-Gly-OH in stead of the wild-type sequence Ala-Pro-Ser-Ser-OH. The introduction of a COOH-terminal or penultimate lysine residue in alphaA-crystallin makes this protein a good substrate for TGC(17) . The eight COOH-terminal residues indeed form a flexible extension from the surface of this homomultimeric protein(23) . Residue Xaa at position 172 in the mutants was occupied by different residues, as presented in Fig. 1, covering the various types of amino acid side chains. The glycine residue behind the introduced amine donor lysine was added because this has been shown to enhance the substrate capacity of the mutant proteins and thus would facilitate analysis(17) . The presence of leucine rather than the wild-type proline before the variable residue Xaa has little influence on the TG reactivity (17) and was chosen because this enabled us to include four mutant clones from our previous series in the present study.

In our previous work we only assessed the reactivity of the mutants toward TGC and used a single biotinylated peptide as the amine acceptor probe. Considering that TGC and factor XIII react to different extents with a particular substrate protein(13, 24) , we now wanted to compare the reactivity of the mutant proteins with both TGC and factor XIII. Binding of the amine acceptor substrate induces conformational changes in the enzyme, creating a suitable binding place for the amine donor protein(10) . It thus can be envisaged that different types of amine acceptor substrates may modulate the binding of the mutated amine donor substrates to different extents. We therefore used two different amine acceptor probes: a biotinylated hexapeptide TVQQEL, as used in our previous work(22) , and a biotinylated heptapeptide PGGQQIV, patterned on the amine acceptor sequence in fibronectin(25) . Finally, to improve the quantitative assessment of the TG reactions, we now used purified mutant alphaA-crystallins rather than crude water-soluble E. coli extracts as in our previous study(17) .

The 13 mutant alphaA-crystallins and the wild-type protein were constructed, expressed, and isolated as described under ``Materials and Methods.'' The nature of the mutations was ascertained by sequencing of the mutant clones. As shown in Fig. 2D, the wild-type and mutant alphaA-crystallins were effectively purified by the employed procedure. Samples of the purified alphaA-crystallins were reacted with TGC and the hexapeptide probe (Fig. 2A), TGC and the heptapeptide probe (Fig. 2B), and factor XIII and the hexapeptide probe (Fig. 2C). Inspection of the resulting streptavidin blots and comparison with the CBB-stained pattern in Fig. 2D immediately reveal conspicuous differences in the reactivity of the various mutants. A quantitative representation of these reactivities was obtained by densitometric screening of the blots (Fig. 3). The obtained values are normalized by relating them to the densities of the corresponding CBB-stained bands, thus correcting for differences in protein concentrations and loading variations. All measurements were performed in duplicate on three independently analyzed samples.


Figure 2: Variation in lysine substrate capacity of mutant alphaA-crystallins for TGC and factor XIII. Streptavidin blots are shown after reaction of the wild-type (wt) and mutant proteins with TGC and biotinylated hexapeptide (A), TGC and biotinylated heptapeptide (B), and factor XIII and biotinylated hexapeptide (C). D gives a corresponding Coomassie Brilliant Blue-stained pattern. The different residues preceding the amine donor lysine in the mutants are indicated by the one-letter notation above and below the lanes. Lane x contains a mutant alphaA-crystallin that is identical to mutant G, apart from the presence of an additional serine at the COOH terminus (Leu-Gly-Lys-Gly-Ser-OH). This mutant was obtained by screening clones obtained with the degenerated linker 3 from (17) .




Figure 3: Comparison of the TG-mediated incorporation of amine acceptor probes in mutant alphaA-crystallins. A, incorporation of biotinylated hexapeptide by TGC (cf.Fig. 2A). B, incorporation of biotinylated heptapeptide by TGC (cf.Fig. 2B). C, incorporation of biotinylated hexapeptide by factor XIII (cf.Fig. 2C). The bars indicate the relation between streptavidin staining and the amount of mutant protein at an arbitrary scale as determined by densitometric scanning. For further details, see ``Materials and Methods.''



Fig. 2and Fig. 3reveal some features that are quite consistent, independent of the probe and type of TG used. The presence of glycine before the amine donor lysine is unfavorable for the cross-linking of mutant and probe, although less so for factor XIII. The aliphatic side chains of alanine, valine, and leucine seem to be favorable, but reactivity does not increase proportionally to the length of the side chain. The TGs seem to be reasonably tolerant to the structure-breaking effect of proline. The size and positive charge of arginine have a positive effect in all instances, but histidine is of intermediate efficacy. The bulky indole side chain of tryptophan is less favorable than the phenyl ring of phenylalanine. The addition of the hydroxyl group in tyrosine slightly decreases the substrate activity. Serine generally makes the mutants good substrates, but compared with alanine the presence of the hydroxyl group variably influences the substrate capacity. Finally, the negative charge of aspartic acid, as compared with the carboxamide group of asparagine, clearly has an adverse effect.

Taken together, it thus appears that both TGC and factor XIII have a broad tolerance toward various types of side chains preceding the amine donor lysine. Apart from glycine and the negative charge of aspartic acid, a broad but variable acceptance is exhibited for aliphatic, aromatic, polar, and positively charged side chains of various sizes. Although differences in reactivity for TGC are measured when using the hexapeptide or heptapeptide probes (Fig. 3, compare A and B), it appears that the overall pattern is remarkably similar. This allows the conclusion that in this system the first substrate does not greatly influence the amine specificity of the TG reaction. The same holds true for the comparison of TGC and factor XIII, although the negative influence of tryptophan is conspicuous for factor XIII (Fig. 3, A and C). However, more detailed kinetic measurements, which we could not perform with our assay, may still reveal more profound differences between the two TGs. Our findings are in agreement with earlier evidence that TGC and factor XIII have very similar amine binding modes(10, 19) . The observation that TGC has a broader substrate specificity than factor XIII (10) seems not simply attributable to different effects of the residue preceding the amine donor lysine.

The present series of mutants is meant to highlight the importance for TG reactivity of the residue directly preceding the substrate lysine in a protein. Although this residue may be the most influential(10, 17, 19) , other nearby residues further modulate the reactivity. As an indication to that end, we included another mutant in lane x in Fig. 2. This mutant only differs from mutant G (Leu-Gly-Lys-Gly-OH) in having an additional COOH-terminal serine (Leu-Gly-Lys-Gly-Ser-OH). It appears that this mutant consistently has an enhanced reactivity as compared with mutant G (Fig. 3). This is in agreement with the suggestion that the active site of TGs corresponds in size to at least 9-10 residues of the polypeptide in their substrates(10) .

How do our observations compare with the information available about the influence of the NH(2)-terminally neighboring residue of amine donor lysines in peptides and proteins? Extensive studies with synthetic peptides have demonstrated that replacing a leucine for a glycine residue in front of the amine donor lysine significantly enhanced the substrate activity toward TGC and factor XIII(10, 19) . Other hydrophobic amino acids in this position might provide a similar effect. Analogs of Xaa-Lys sequences, where Xaa is Ala, Phe, Leu, Val, or Trp, indeed all denoted high specificity in the TG reaction(20) . This led to the idea that the major force in acyl enzyme-lysine peptide interaction is provided by the secondary hydrophobic binding through the side chain of the residue adjacent to and on the amino side of the substrate lysine(10, 19) . Our observations are in agreement with a positive effect of various hydrophobic residues. However, they also indicate that under our conditions, basic and uncharged polar side chains in front of the amine donor lysine can be equally effective in directing its proper orientation in the active site of TGs and hence in determining its cross-linking capacity.

Only a very small number of amine donor lysines have been characterized in substrate proteins for TGs. Fourteen identified substrate lysines for TGC(26, 27, 28, 29, 30, 31, 32) have recently been tabulated(17) . Another lysine substrate for TGC has since been identified in Alzheimer amyloid protein betaA4, located in the sequence -SNKG-(33) . For factor XIII the substrate lysine in the fibrin chain has been characterized long ago (-GAKQ-)(34) . Hohl et al.(35) were the first to isolate isodipeptides of an in situ cross-linked protein, (i.e. loricrin from the cornified cell envelope). This revealed the internal (-SVKY-) and the COOH-terminal (-PSK) lysine residues as substrates for the epidermal transglutaminases. Recently, Steinert and Marekov (18) identified another 12 cross-linked lysine residues from the cornified envelope. These involved the sequences -YQKKQ- and -QQKQ- in loricrin, -QQKQ- and -KQK in SPR1, -KSK in SPR2, -QDKVKA- and -PVKG- in prepro-elafin, and -GSKS-, -GTKS-, and -SSKQ- in keratins 1, 2e, and 10, respectively. Among this total of 30 characterized amine donor residues, the residues directly preceding the substrate lysines are Gln (6 times), Lys (5 times), Ser (5 times), Ala (3 times), Thr (3 times), Val (3 times), Arg (2 times), Leu (1 time), Asn (1 time), and Asp (1 time). This listing combines results from various TGs and from a limited number of proteins but nevertheless suggests a preference for uncharged and basic polar residues, as well as for the smaller aliphatic ones. It is noteworthy that the residues found in our studies to negatively influence TG reactivity, like Asp, Gly, Pro, His, and Trp, indeed appear to be largely avoided. These data thus are in accord with the preferences that we established for our mutant proteins.

Most of the current insight in the basis for specificity of TG-catalyzed isodipeptide bond formation stems from the work of Folk and co-workers on small model substrates(10, 19, 20) . It has been emphasized (10) that little has been learned yet about the manner in which the TGs operate on macromolecular substrates in biological systems. Using site-directed mutagenesis, we now show that the residue preceding an accessible amine donor lysine in a native protein has considerable influence on TG cross-linking potential. This corroborates and extends the results of earlier work on synthetic peptides(19, 20) . Further studies are required to reveal the possible modulating effects of native protein conformation in comparison with small model substrates on substrate specificity and mechanism of action of TGs.


FOOTNOTES

*
This work was supported by the Netherlands Organization for Scientific Research. 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.: 31-80-616848 or 31-80-614254; Fax: 31-80-540525.

(^1)
The abbreviations used are: TG, transglutaminase (protein-glutamine:amine -glutamyltransferase, EC 2.3.2.13); CBB, Coomassie Brilliant Blue.


REFERENCES

  1. Greenberg, C. S., Birckbirchler, P. J., and Rice, R. H. (1991) FASEB J. 5,3071-3077 [Abstract/Free Full Text]
  2. Hand, D., Perry, M. J. M., and Haynes, L. W. (1993) Int. J. Dev. Neurosci. 11,709-720 [CrossRef][Medline] [Order article via Infotrieve]
  3. Aeschlimann, D., and Paulsson, M. (1994) Thromb. Haemostasis 71,402-415 [Medline] [Order article via Infotrieve]
  4. Kim, I.-G., Lee, S. C., Lee, J. H., Yang, J. M., Chung, S. I., and Steinert, P. M. (1994) J. Invest. Dermatol. 103,137-142 [Abstract]
  5. Fesus, L., Davies, P. J. A., and Piacenti, M. (1991) Eur. J. Cell Biol. 56,170-177 [Medline] [Order article via Infotrieve]
  6. Knight, C. R. L., Rees, R. C., and Griffin, M. (1991) Biochim. Biophys. Acta 1096,312-318 [Medline] [Order article via Infotrieve]
  7. Cordella-Miele, E., Miele, L., and Mukherjee, A. B. (1990) J. Biol. Chem. 265,17180-17188 [Abstract/Free Full Text]
  8. Eitan, S., Solomon, A., Lavie, V., Yoles, E., Hirschberg, D. L., Belkin, M., and Schwartz, M. (1994) Science 264,1764-1768 [Medline] [Order article via Infotrieve]
  9. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M.-J., and Graham, R. M. (1994) Science 264,1593-1596 [Medline] [Order article via Infotrieve]
  10. Folk, J. E. (1983) Adv. Enzymol. 54,1-56 [Medline] [Order article via Infotrieve]
  11. Lorand, L., and Conrad, S. M. (1984) Mol. Cell Biochem. 58,9-35 [Medline] [Order article via Infotrieve]
  12. Yee, V. C., Pedersen, L. C., Le Trong, I., Bishop, P. D., Stenkamp, R. E., and Teller, D. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,7296-7300 [Abstract]
  13. Gorman, J. J., and Folk, J. E. (1984) J. Biol. Chem. 259,9007-9010 [Abstract/Free Full Text]
  14. Aeschlimann, D., Paulsson, M., and Mann, K. (1992) J. Biol. Chem. 267,11316-11321 [Abstract/Free Full Text]
  15. Coussons, P. J., Price, N. C., Kelly, S. M., Smith, B., and Sawyer, L. (1992) Biochem. J. 282,929-930 [Medline] [Order article via Infotrieve]
  16. Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269,28309-28313 [Abstract/Free Full Text]
  17. Groenen, P. J. T. A., Smulders, R. H. P. H., Peters, R. F. R., Grootjans, J. J., van den IJssel, P. R. L. A., Bloemendal, H., and de Jong, W. W. (1994) Eur. J. Biochem. 220,795-799 [Abstract]
  18. Steinert, P. M., and Marekov, L. N. (1995) J. Biol. Chem. 270,24916-24925
  19. Gross, M., Whetzel, N. K., and Folk, J. E. (1977) J. Biol. Chem. 252,3752-3759 [Medline] [Order article via Infotrieve]
  20. Schrode, J., and Folk, J. E. (1979) J. Biol. Chem. 254,653-661 [Medline] [Order article via Infotrieve]
  21. Merck, K. B., de Haard-Hoekman, W. A., Oude Essink, B. B., Bloemendal, H., and de Jong, W. W. (1992) Biochim. Biophys. Acta 1130,267-276 [Medline] [Order article via Infotrieve]
  22. Groenen, P. J. T. A., Bloemendal, H., and de Jong, W. W. (1992) Eur. J. Biochem. 205,671-674 [Abstract]
  23. Carver, J. A., Aquilina, J. A., Truscott, R. J. W., and Ralston, G. B. (1992) FEBS Lett. 311,143-149 [CrossRef][Medline] [Order article via Infotrieve]
  24. Shainoff, J. R., Urbanic, D. A., and Di Bello, P. M. (1991) J. Biol. Chem. 266,6429-6437 [Abstract/Free Full Text]
  25. Lorand, L., Parameswaran, K. N., and P. T. Velasco (1991) Proc. Natl. Acad. Sci. U. S. A. 88,82-83 [Abstract]
  26. Pucci, P., Malorni, A., Marino, G., Metafora, S., Esposito, C., and Porta, R. (1988) Biochem. Biophys. Res. Commun. 154,737-740
  27. Porta, R., Esposito, C., Metafora, S., Malorni, A., Pucci, P., Siciliano, R., and Marino, G. (1991) Biochemistry 30,3114-3120 [Medline] [Order article via Infotrieve]
  28. Lorand, L., Velasco, P. T., Murthy, S. N. P., Wilson, J., and Parameswaran, K. N. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,11161-11163 [Abstract]
  29. Merck, K. B., Groenen, P. J. T. A., Voorter, C. E. M., de Haard-Hoekman, W. A., Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. 268,1046-1052 [Abstract/Free Full Text]
  30. Ikura, K., Takahata, K., and Sasaki, R. (1993) FEBS Lett. 326,109-111 [CrossRef][Medline] [Order article via Infotrieve]
  31. Mariniello, L., Esposito, C., Di Pierro, P., Cozzolino, A., Pucci, P., and Porta, R. (1993) Eur. J. Biochem. 215,99-104 [Abstract]
  32. Groenen, P. J. T. A., Grootjans, J. J., Lubsen, N. H., Bloemendal, H., and de Jong, W. W. (1994) J. Biol. Chem. 269,831-833 [Abstract/Free Full Text]
  33. Rasmussen, L. K., Sorensen, E. S., Petersen, T. E., Gliemann, J., and Jensen, P. H. (1994) FEBS Lett. 338,161-166 [CrossRef][Medline] [Order article via Infotrieve]
  34. Chen, R., and Doolittle, R. F. (1971) Biochemistry 10,4486-4491
  35. Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R., and Steinert, P. M. (1991) J. Biol. Chem. 266,6626-6636 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.