Document Detail


Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein.
MedLine Citation:
PMID:  8318003     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The V3 loop of the HIV (human immunodeficiency virus)-1 envelope glycoprotein gp120 likely plays a role in HIV-1 infectivity. Although the amino acid sequence of the V3 loop is hypervariable, it contains a conserved region, Gly-Pro-Gly-Arg, that shows similarity to the active-site Gly-Pro-Cys-Arg sequence of inter-alpha-trypsin and trypstatin proteinase inhibitors. The purpose of the present work was to identify proteinases recognizing substrates with basic amino acids in the P1 substrate site that are present in MOLT-4 cells, a human CD4-positive T helper lymphocyte cell line, and to characterize these enzymes in terms of substrate, pH and ionic-strength preferences, size and susceptibility to various inhibitors, including 24- and 36-amino-acid-long V3 loop peptides. Extraction of MOLT-4 cells at low ionic strength solubilized nearly all of the trypsin-like activity, which was separable into five peaks of activity by chromatography on Mono-Q: Peaks 1, 2a, 2b, 3 and 4. All showed a neutral pH optimum, and all except Peak 4 showed optimal activity at high ionic strength. Peak 1 preferred Tos-Gly-Pro-Arg, p-nitroanilide (-pNA) substrate; Peaks 2-4 preferred benzyloxycarbonyl-Val-Leu-Gly-Arg-pNA. Peak 1, a zinc-dependent enzyme with serine and histidine in the active site, exhibited an M(r) of 75,000 on Superose 12 and was poorly inhibited by V3 loop peptides. Peak 2 contained two overlapping peaks, called 2a and 2b, that exhibited properties of zinc-dependent metalloproteinases. Gel filtration of Peak 2 activities revealed a major peak of activity at 81 kDa and a shoulder centred at 240 kDa. Each was modestly inhibited by V3 loop peptides. Peak 3, a zinc-dependent proteinase, exhibited a molecular mass of 100 kDa by gel filtration and was particularly sensitive to inhibition by V3 loop peptides. Peak 4 exhibited a molecular mass of 1100 kDa by gel filtration and was not inhibited by V3 loop peptides. None of these enzymes could be classified as mast-cell tryptase, and material in MOLT-4 cells cross-reactive with anti-(human tryptase) antibodies was not detected. Whether any of the MOLT-4 proteinases described in this study play a role in HIV-1 infectivity remains to be examined.
Authors:
I T Harvima; R J Harvima; G Nilsson; L Ivanoff; L B Schwartz
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Biochemical journal     Volume:  292 ( Pt 3)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1993 Jun 
Date Detail:
Created Date:  1993-07-27     Completed Date:  1993-07-27     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  711-8     Citation Subset:  IM; X    
Affiliation:
Department of Internal Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acids, Diamino*
Chromatography, Gel
Chromatography, Ion Exchange
Conserved Sequence
Endopeptidases / isolation & purification*,  metabolism*
HIV Envelope Protein gp120 / metabolism
HIV-1 / pathogenicity
Humans
Kinetics
Molecular Sequence Data
Oligopeptides / pharmacology
Peptide Fragments / metabolism,  pharmacology
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Protease Inhibitors / pharmacology
Substrate Specificity
T-Lymphocytes
Tumor Cells, Cultured
Grant Support
ID/Acronym/Agency:
AI-20487/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids, Diamino; 0/HIV Envelope Protein gp120; 0/Oligopeptides; 0/Peptide Fragments; 0/Protease Inhibitors; EC 3.4.-/Endopeptidases
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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