From the Department of Pathology, Immunology and
Laboratory Medicine, Center for Mammalian Genetics and Diabetes Center
of Excellence, College of Medicine, and ¶ Department of Medicinal
Chemistry, College of Pharmacy and The McKnight Brain Institute,
University of Florida, Gainesville, Florida 32610
Received for publication, December 21, 2000, and in revised form, February 12, 2001
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ABSTRACT |
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Here we describe the cloning, localization, and
characterization of a novel mammalian endo-apyrase (LALP1)
in human and mouse. The predicted human LALP1 gene encodes
a 604-amino acid protein, whereas the mouse Lalp1 gene
encodes a 606-amino acid protein. The human and mouse genes have 88%
amino acid sequence identity. These genes share considerable homologies
with hLALP70, a recently discovered mammalian lysosomal endo-apyrase.
The human LALP1 gene resides on chromosome 10q23-q24 and
contains 12 exons and 11 introns covering a genomic region of ~46
kilobase pairs. The subcellular localization and enzymatic
activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with
substrate preference for nucleoside triphosphates UTP, GTP, and CTP.
Apyrases are hydrolytic enzymes that cleave nucleoside
tri- and diphosphates in a calcium- or magnesium-dependent
manner but are insensitive to P-, F-, or V-type ATPase inhibitors (1). Both apyrases and ATPases can use ATP as substrates; however, there are
substantial differences (1, 2). For instance, the ATPases are highly
selective toward their substrate ATP with ADP and phosphate as reaction
products. On the contrary, apyrases can use different nucleoside tri-
and diphosphates as their substrates, with phosphates and nucleoside
monophosphates as the main reaction products. Additionally, sequence
comparison among various apyrases revealed the presence of apyrase
conserved regions, which are highly conserved among all apyrases from
various organisms such as plants, parasites, and mammals. Thus far, two
types of apyrases, ecto-apyrases and endo-apyrases, have been described
(2). Ecto-apyrases such as CD39 are apyrase enzymes with their
catalytic domain exposed on the cell surface (1-3) and are believed to
be involved in many processes such as neurotransmission (4), platelet
aggregation, and blood pressure regulation (5). On the other hand,
endo-apyrases such as uridine diphosphatase and the 700-kDa lysosomal
apyrase-like protein (LALP70) are apyrase enzymes with their catalytic
domain localized intracellularly (6, 7). The biological functions of
the endo-apyrases are unknown, although it was suggested that they are
important for regulating the level of activated sugar during protein glycosylation.
Recently, Wang and Guidotti (6) identified the first mammalian
endo-apyrase, human uridine diphosphatase. This enzyme is predicted to
be a 610-amino acid protein with two putative transmembrane domains.
Using a myc-tagged version of this protein, this enzyme was found to be
in the luminal side of the Golgi apparatus and had the highest
catalytic activity using UDP as its substrate and had lower activities
using GDP, CDP, UTP, GTP, CTP, and TTP. Interestingly,
Biederbick et al. (7) reported altered substrate preferences of LALP70, which is a splicing variant of uridine diphosphatase and has an additional 8 amino acids (VSFASSQQ) resulting from the inclusion of an alternate exon. The enzymatic properties of
this splice variant revealed a broad substrate specificity, with CTP,
UDP, CDP, GTP, and GDP as preferred substrates. Using antibodies and a
green fluorescent protein-tagged version of this splicing variant,
LALP70 was co-localized with the autophagic marker monodansylcadaverine
and the lysosomal protein lamp1, suggesting a lysosomal/autophagic
vacuole subcellular location (8). Although most of the transiently
expressed LALP70/GFP1 fusion
protein was co-localized with lamp1-positive vacuoles, an association
with the Golgi apparatus and the endoplasmic reticulum could not be
ruled out. These studies clearly demonstrated that LALP70/uridine
diphosphatase is the first mammalian member of the endo-apyrase gene
family. Here we report the molecular cloning and characterization of
the LALP1 gene, which closely resembles LALP70 in
both structure and enzymatic property. Hence, we propose that
LALP1 is a second member of the endo-apyrase gene family.
5'-RACE and 3'-RACE--
5'- and 3'-RACE reactions were
conducted with the SMART RACE kit (CLONTECH)
according to the manufacturer's instructions, with a slight
modification as described previously (11, 12). Briefly, for 5'-RACE,
the first-strain cDNA synthesis is primed using a gene-specific
primer and a SMART oligonucleotide with human brain total RNA.
After reverse transcription reaches the end of the mRNA template,
several dC residues are added to the end of the cDNA. The SMART
oligonucleotide, which contains several 3-dG at its 3' end, anneals to
the tail of the newly synthesized cDNA and then serves as a
template for further extension of the cDNA by RT. After the RT
reaction, an internal gene-specific reverse primer and a UP
primer, which is complementary to SMART oligonucleotide, were used to
perform PCR using the RT products as templates. To increase the
specificity and product yield of 5'-RACE, nested PCR was then performed
using another internal gene-specific primer and NUP primer
(internal primer of UP primer). For 3'-RACE, the first-strand cDNA
was synthesized using a modified oligo(dT) with a UP oligonucleotide tail.
The UP primer and a gene-specific forward primer were used for
first-round PCR. Nested PCR was performed using NUP and an internal gene-specific forward primer. PCRs were carried out in a final
volume of 20 µl. After the RT-PCR, the samples were denatured at
94 °C for 5 min; amplifications were carried out with 5 cycles of
30-s denaturing at 94 °C, 30-s annealing at 68 °C, and a
4-min extension at 72 °C followed by 30 cycles of 30-s denaturing at 94 °C, 30-s annealing at 62 °C, and a 4-min extension at
72 °C. PCR products obtained from nested PCR were loaded onto a 2%
agarose gel, and individual bands were excised from the gel for
direct sequencing. The PCR product was subcloned into TA vector
(Invitrogen, San Diego, CA), and the sequence was determined with ABI
310 DNA sequencer as described previously (12-14).
Expression Analysis by Competitive RT-PCR--
Total
RNA was isolated from various mouse tissues using the RNeasy kit
(Qiagen) according to the manufacturer's instructions. Total RNA (5 µg) was used for RT reactions in a total volume of 20 µl using a
poly(T)-plus primer as described previously (13, 14). Denatured
RNA samples and the primer were incubated with reverse transcriptase at
42 °C for 1 h. The RT reaction was then stopped by heating the
samples at 95 °C for 10 min. 2 µl of the cDNA was used as
template for a subsequent PCR in a total volume of 20 µl. To assess
the relative expression level of the mouse Lalp1 gene,
competitive RT-PCR was performed. Briefly, a 750-bp Lalp1
fragment (mExon8F and 5'UTR-R primers) was co-amplified with a 450-bp
Expression Constructs--
Two expression constructs were made
to study the enzymatic activity and cellular localization of the human
LALP1 protein. First, full-length LALP1 cDNA was
subcloned into mammalian expression vector pEGFP N1
(CLONTECH) using engineered HindIII
restriction endonuclease sites via PCR. This construct is designated
pEGFP/LALP1. In the second construct (pEGFP/LALP1-GFP), LALP1 cDNA
is fused with a C-terminal GFP to produce LALP1-GFP fusion protein.
Cell Culture and Transfection--
HEK297 cells were cultivated
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum and 50 µg/ml gentamycin and incubated at 37 °C in a
humidified chamber equilibrated with 5% CO2. Cells were
transfected with mammalian pEGFP expression constructs using
LipofectAMINE (Life Technologies, Inc.) as transfection reagent
following the manufacturer's instructions. Transfected cells were
harvested for the apyrase assay 24 h after transfection.
Subcellular Localization with GFP Fusion--
The cellular
localization of LALP1 was determined by transfection experiments with
the LALP1-GFP plasmid. After transfection, cells were cultured in
chamber slides (LAB-TEK) for 12-18 h. Transfected cells were then
fixed with 4% paraformaldehyde for 20 min and mounted with coverslips.
The fluorescent images were obtained using a confocal microscope
(Bio-Rad) equipped with an argon laser with excitation at 488 nm and
detection at 510-530 nm bandpass for GFP.
Nucleotide Phosphatase Activity Assay--
Nucleotide
phosphatase activity was measured in crude membranes from HEK293 cells
transfected with pEGFP vector or pEGFP/LALP1 construct. Ten flasks
(T75) of transfected HEK293 cells were homogenized with a Dounce
homogenizer, and nuclei were separated according to the method of Wang
and Guidotti (6). To separate the crude membrane fraction from the
cytosol, the postnuclear supernatant was centrifuged at 100,000 × g. The pellets were resuspended in 500 µl of 20 mM Hepes, pH 7.4, 120 mM NaCl, 5 mM
KCl, and 0.2 mM EDTA containing 0.1% Triton X-100. The
protein concentration in each sample was determined with bicinchoninic
acid (Pierce) according to the manufacturer's instructions.
To measure apyrase activity, HEK297 membrane suspension containing 10 µg of total protein was adjusted to 45 µl with reaction buffer
containing 20 mM Hepes, pH 7.4, 120 mM NaCl, 5 mM KCl, 0.2 mM EDTA, 1 mM
NaN3, and 0.5 mM
Na3VO4, with or without 5 mM CaCl2. After preincubation for 5 min at 37 °C nucleotide
phosphatase reactions were initiated by the addition of 5 µl of the
same buffer containing 10 mM nucleotide phosphate
substrates to give a final concentration of 1 mM. Samples
were incubated for 30 min at 37 °C. NTP/NDP hydrolysis under these
conditions was linear up to 30 min. Apyrase activity was determined by
measuring the inorganic phosphate released as described previously
(6-7). Values obtained from samples without CaCl2 or
MgCl2 were subtracted from those obtained with
CaCl2 or MgCl2. All measurements were done in
triplicate. Student's t test was performed to assess
statistical significance.
Cloning of the Human LALP1Gene--
The human
LALP1 gene was cloned using SMART RACE technology. Based on
the sequence of expressed sequence tag N35618 in the human
chromosomal region 10q23-q24 (9, 10), several gene-specific reverse
primers (hLALP1-RT, hLALP1-R1, and hLALP1-R2) and forward primers
(hLALP1-F1 and hLALP1-F2) were designed for 5'-RACE and 3'-RACE,
respectively. The resulting full-length sequence (Fig. 1) was 2962 bp (GenBankTM
accession number AF269255). A GenBankTM search indicated
that the human LALP1 gene is contained in clone RP11-483F11
(GenBankTM accession number AL133353). Comparison of
cDNA and genomic sequences suggested that LALP1 has 12 exons that expand about 46 kilobase pairs of genomic sequence
(Fig. 2). The cloned cDNA coincides
almost exactly with the computer-predicted structure. The translation
initiation codon ATG is located near the end of exon 1, which contains
only eight coding nucleotides. Multiple in-frame stop codons are
present upstream of the putative first ATG. A CpG island was identified
at Cloning of the Mouse Lalp1Gene--
The mouse
Lalp1 gene was cloned based on homology with its human
homologue. Briefly, two human LALP1 primers (hExon4-F and hExon8-R) were used to amplify the mouse gene from mouse brain cDNA. A fragment of 450 bp encompassing the region from exon 4 to
exon 8 was obtained and directly sequenced. This mouse fragment shares
86.8% homology with the human LALP1 homologous region, confirming that the fragment is indeed derived from the mouse Lalp1 gene. A mouse reverse primer (mLALP-R1) was designed
based on the partial mouse Lalp1 sequence and used together
with a human forward primer (hExon1-F) in RT-PCR. This RT-PCR generated
a fragment of 730 bp, which shares 89.5% homology with the human
LALP1 sequence. To obtain the complete sequences at the 5'
and 3' ends of the gene, 5'- and 3'-RACE were performed using several
reverse and forward primers (mLALP1-RT, mLALP1-R2, mLALP1-F1, and
mLALP1-F2).
Mouse Lalp1 cDNA contains 3268 bp (GenBankTM
accession number AF288221) with an open reading frame of 1818 bp and
encodes a protein of 606 amino acids (with a molecular mass of 69.99 kDa). The mouse and human cDNA sequences share 87% nucleotide
sequence similarity in the entire coding region, whereas the sequence
homology in 5'-UTR and 3'-UTR is poor, further supporting the open
reading frame assignment. The deduced protein sequences are also highly conserved between human and mouse (88% identity).
To compare the relative expression levels in different
tissues, competitive RT-PCR was performed with RNA from different mouse tissues. As shown in Fig. 3,
Lalp1 is highly expressed in most tissues including the
brain, kidney, liver, and testis. The expression is relatively lower in
the lung, thymus, and heart. Among all tissues analyzed, the expression
is lowest in the spleen. These results suggest that the LALP1 protein
must play critical roles in the cellular functions of many different
tissues.
Nucleotide Phosphatase Activities--
A BLAST search
revealed that the LALP1 and Lalp1 proteins have significant sequence
homologies with human LALP70 and guanosine diphosphatase protein family
members (Fig. 4). The human LALP1 shares
59% identity and 71% similarity to LALP70, 58% identity and 70%
similarity to guanosine diphosphatase, and 25% identity and 42%
similarity to CD39. These results suggest that LALP1 may be an
apyrase.
To determine whether LALP1 is indeed an apyrase, nucleotide
phosphotase activity was measured in HEK293 cells transfected with the
pEGFP vector or the full-length LALP1 cDNA constructs (pEGFP/LALP1). The experiments were done in the presence of 1 mM azide (inhibitor of F-type ATPase) and 0.5 mM vanadate (inhibitor of P-type ATPase), which do not
inhibit the activities of E-ATPases (1-2). As shown in Fig.
5, the mean activities were higher for cells containing LALP1 expression vector than in cells containing control vector. For example, the CTPase activity (24.9 ± 1.46 nmol Pi/min/mg) with the LALP1 construct is 5-fold higher
than the CTPase activity (4.34 ± 2.02 nmol Pi/min/mg)
for cells transfected with control vector. Student's t test
suggested that the mean activities were significantly different between
LALP1 and control vector with UTP (p < 0.02), GTP
(p < 0.002), and CTP (p < 0.0001) as
the substrates (Fig. 5). Furthermore, the nucleotide phosphatase activities are absolutely dependent on the presence of Ca2+
ions (data not shown). Therefore, our data indicate that human LALP1 is
truly an apyrase with a substrate preference for UTP, GTP, and CTP. It
is interesting to note that the HEK293 cells have very high enzymatic
activities for the GDP and UDP nucleotide substrates (controls in Fig.
5) due to endogenous apyrases. However, GDP and UDP do not seem to be
preferred substrates for the LALP1 protein because LALP1-overexpressing
cells do not have significantly higher enzymatic activities compared
with control cells.
We then determined whether LALP1 is an ecto-apyrase or an
endo-apyrase. Two sets of experiments were performed. First, the enzymatic activity of intact and disrupted cells was compared to
determine whether it is located intracellularly or extracellularly. The
CTPase activity of LALP1 cDNA-transfected HEK293 cells
increased significantly after the cells were treated with 0.1% Triton
X-100 or disrupted by mechanical forces (data not shown). These results suggest that human LALP1 is located inside the cell.
Second, we determined the cellular localization of LALP1
using a LALP1-GFP fusion construct. The expression of LALP1-GFP fusion protein could be clearly observed as a punctuate distribution pattern
under a confocal fluorescence microscope (Fig.
6A), indicating an
intracellular vesicular compartmentalization as compared with a typical
soluble GFP intracellular distribution (Fig. 6B). However, we did not observe any significant plasma membrane fluorescence, even
under prolonged transfection conditions. Therefore, the LALP1-GFP fusion protein was associated with intracellular membrane compartments instead of the plasma membrane. This result further supports the notion
that LALP1 is an endo-apyrase.
In conclusion, we have cloned the human LALP1 and mouse
Lalp1 genes that share a high degree of sequence homology
with mammalian endo-apyrases. Studies of the enzymatic activities and
intracellular localization strongly suggest that these genes encode a
novel member of the mammalian endo-apyrase gene family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-actin fragment (Table I) in the same
PCR. The annealing temperature was set to 58 °C, and the PCR was run
for 30 cycles. The PCR products were analyzed on a 2% agarose gel in
Tris-borate EDTA buffer.
Primers used in this study
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
124 bp upstream of the putative first ATG. A putative
polyadenylation signal sequence (AATAAA) is found at 28 bp upstream of
the poly(A) tail. The LALP1 gene contains 1,812 nucleotides
of coding sequence and encodes a protein of 604 amino acids with a
molecular mass of 69.88 kDa. Interestingly, the exon/intron structure
of LALP1 is almost identical to that of the
LALP70 gene (7).
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Fig. 1.
Nucleotide and protein sequences of the human
LALP1. The four highly conserved apyrase regions
(ACR1-4) are in italic boldface type. The putative
membrane-spanning regions determined using the algorithm of Kyte and
Doolittle (15) are underlined.
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Fig. 2.
Genomic strucuture of the human
LALP1 gene. Black boxes represent
exons, whereas the lines represent introns.
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Fig. 3.
Tissue distribution of Lalp1
in mice. RT-PCR was conducted using total RNA from various
mouse tissues and the mExon8-F/5'UTR-R primer pair (752 bp). A 450-bp
-actin fragment was co-amplified in the same PCR. PCR products were
electrophoresed on a 2% agarose gel. The ratio of
Lalp1/
-actin reflects the relative expression level of
the Lalp1 gene.
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Fig. 4.
Protein sequence alignment of apyrases.
The sequences aligned include hLALP1 (human LALP1), mLALP1 (mouse
Lalp1), hLALP70 (human LALP70 from AJ246165), hCD39 (human CD39
from NP_001767), and yGDA (yeast guanosine diphosphatase from
NP_010872). Identical amino acids are indicated by black
boxes, whereas conserved amino acids are highlighted in
gray. Deletions are represented by a dash.
Accession numbers are from GenBankTM.
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Fig. 5.
Nucleotide phosphatase activity of human
LALP1. Activity for each of the eight nucleotides was assayed with
crude membrane preparations from cells transfected with the LALP1
cDNA construct (pEGFP/LALP1) ( ) and the same control vector
without a LALP1 insert (pEGFP) (
). The difference in activities
between the LALP1 construct and control vector represents
the increased activity of the transfected LALP1 expression
vector. Activities for each of the eight nucleotides and each of the
two cell lines were assayed in three independent experiments. The mean
and S.D. of the three experiments are presented.
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Fig. 6.
Subcellular localization of human
LALP1 using LALP1-GFP fusion
protein. Full-length human LALP1 cDNA is
fused with GFP at its C terminus and transfected into HEK293 cells. The
protein localization was examined 18 h after transfection.
A, LALP1-GFP; B, GFP.
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ACKNOWLEDGEMENTS |
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We thank James Yang for performing statistical analyses and Drs. Predeep G. Kumar and Malini Laloraya for helpful discussion.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grants 1R01DK53266-01 (to J.-X. S) and AG015688 (to D. H. W).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Supported by a University of Florida Alumni Fellowship.
** To whom correspondence should be addressed: Dept. of Pathology, Immunology and Laboratory Medicine, Box 100275, University of Florida, Gainesville, FL 32610. Tel.: 352-392-0677; Fax: 352-392-3053; E-mail: she@ufl.edu.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M011569200
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescent protein; RACE, rapid amplification of cDNA ends; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); UTR, untranslated region.
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REFERENCES |
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---|
1. | Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214[Medline] [Order article via Infotrieve] |
2. | Komoszynski, M., and Wojtczak, A. (1996) Biochim. Biophys. Acta 1310, 233-241[Medline] [Order article via Infotrieve] |
3. |
Kaczmarek, E.,
Koziak, K.,
Sevigny, J.,
Siegel, J. B.,
Anrather, J.,
Beaudoin, A. R,
Bach, F. H.,
and Robson, S. C.
(1996)
J. Biol. Chem.
271,
33116-33122 |
4. |
Marcus, A. J.,
Broekman, M. J.,
Drosopoulos, J. H. G.,
Islam, N.,
Alyonycheva, T. N.,
Safier, L. B.,
Hajiar, K. A.,
Posnett, D. N.,
Schoenborn, M. A.,
Schooley, R. B.,
Gayle, R. B.,
and Maliszewski, C. R.
(1997)
J. Clin. Invest.
99,
1351-1360 |
5. | Zimmermann, H. (1996) Biochem. J. 285, 345-365 |
6. |
Wang, T. F.,
and Guidotti, G.
(1998)
J. Biol. Chem.
273,
11392-11399 |
7. |
Biederbick, A.,
Kosan, C.,
Kunz, J.,
and Elsasser, H. P.
(2000)
J. Biol. Chem.
275,
19018-19024 |
8. |
Biederbick, A.,
Rose, S.,
and Elsasser, H. P.
(1999)
J. Cell Sci.
112,
2473-2484 |
9. | Wang, C. Y., Shi, J. D., Huang, Y. Q., Cruz, P. E., Ochoa, B., Bobbilynn, H.-L., Semiromi, A. D., and She, J. X. (1999) Genomics 60, 12-19[CrossRef][Medline] [Order article via Infotrieve] |
10. | Wang, C. Y., Huang, Y. Q., Shi, J. D., Marron, M. P., Ruan, Q. G., Ochoa, B., Bobbilynn, H. L., and She, J. X. (1999) Am. J. Med. Genet. 84, 454-459[CrossRef][Medline] [Order article via Infotrieve] |
11. | Li, Q. Z., Wang, C. Y., Shi, J. D., Ruan, Q. G., Cruz, P. E., Eckenrode, S., Semiromi, A. D., and She, J. X. (2001) Genomics, in press |
12. | Wang, C. Y., Semiromi, A. D., Huang, W., Connor, E., Shi, J. D., and She, J. X. (1998) Hum. Genet. 103, 681-685[CrossRef][Medline] [Order article via Infotrieve] |
13. | Wang, C. Y., Shi, J. D., Semiromi, A. D., and She, J. X. (1999) Genomics 55, 322-326[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ruan, Q. G., Wang, C. Y., Shi, J. D., and She, J. X. (1999) Autoimmunity 13, 307-313[CrossRef] |
15. | Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |