ABSTRACT
Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.
Subject(s)
GTP-Binding Proteins/agonists , GTP-Binding Proteins/metabolism , Receptors, Cell Surface/agonists , Receptors, G-Protein-Coupled , Urotensins/pharmacology , Vasoconstrictor Agents/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Calcium/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , GTP-Binding Proteins/genetics , Humans , Macaca fascicularis , Male , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Sequence Homology, Amino Acid , Tissue Distribution , Urotensins/metabolism , Vasoconstrictor Agents/metabolismABSTRACT
High levels of active HIV-1 protease (PR) were produced in Escherichia coli, amounting to 8-10% of total cell protein. High production levels were achieved by altering the following parameters: (1) codon preference of the coding region, (2) A+T-richness at the 5' end of the coding region, and (3) promoter. To circumvent the toxicity of HIV-1 PR in E. coli, the gene was expressed as a fusion protein with two different proteolytic autocleavage sequences. In both the cases, the fusion protein could be cleaved in vivo to give an active molecule with the native sequence at the N terminus.
Subject(s)
Escherichia coli/genetics , HIV Protease/biosynthesis , HIV-1/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular/methods , DNA, Recombinant , Electrophoresis, Polyacrylamide Gel , Gene Expression , Genes, Synthetic , HIV Protease/genetics , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sequence Homology, Nucleic AcidABSTRACT
Synthetic peptides based on functionally equivalent (as defined by similar patterns of chemically equivalent amino acids) serine protease inhibitor (serpin) C-terminal sequences inhibit both classical and alternative pathways of complement activation. Inhibition was also found with hybrid peptides consisting of the cleavage site of one serpin (antithrombin III, alpha-1-antitrypsin, or antichymotrypsin) attached to the short and long functionally equivalent protease binding cores of the other two serpins. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to the long binding core of antithrombin III was selective in inhibiting the classical pathway with no effect on the alternative pathway at a concn of 10 microM. Extension of the functional equivalence hypothesis has produced inhibitors of complement activation named generic and generic +, whose sequences differ by 77% or 87%, respectively, from those of all three serpin sequences. A hybrid peptide composed of the antithrombin III cleavage site attached to the generic peptide is an inhibitor of complement activation at 500 nM, the most potent inhibitor found in this study.
Subject(s)
Complement Activation/drug effects , Serpins/chemistry , Amino Acid Sequence , Antithrombin III/chemistry , Complement Hemolytic Activity Assay , Humans , In Vitro Techniques , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Serpins/chemical synthesis , Structure-Activity Relationship , alpha 1-Antichymotrypsin/chemistry , alpha 1-Antitrypsin/chemistryABSTRACT
Formation of the covalently stabilized complex of alpha 1-antitrypsin (alpha 1-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the alpha 1-AT molecule and hydrolysis of a reactive-site peptide bond. An approximately 4-kDa carboxyl-terminal cleavage fragment is generated. alpha 1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the alpha 1-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the alpha 1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of alpha 1-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of alpha 1-AT as a candidate ligand (carboxyl-terminal fragment, amino acids 359-374). We show here that synthetic peptides based on the sequence of this region bind specifically and saturably to human hepatoma cells and human monocytes (Kd = 4.0 X 10(-8) M, 4.5 X 10(5) plasma membrane receptors per cell) and mediate increases in synthesis of alpha 1-AT. Binding of peptide 105Y (Ser-Ile-Pro-Pro-Glu-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Tyr-Leu-Ile) is blocked by alpha 1-AT-elastase complexes, antithrombin III (AT III)-thrombin complexes, alpha 1-antichymotrypsin (alpha 1-ACT)-cathepsin G complexes, and, to a lesser extent, complement component C1 inhibitor-C1s complexes, but not by the corresponding native proteins. Binding of peptide 105Y is also blocked by peptides with sequence corresponding to carboxy-terminal fragments of the serpins AT III and alpha 1-ACT, but not by peptides having the sequence of the extreme amino terminus of alpha 1-AT. The results also show that peptide 105Y inhibits binding of 125I-labeled alpha 1-AT-elastase complexes. Thus, these studies demonstrate an abundant, relatively high-affinity cell surface receptor which recognizes serpin-enzyme complexes (SEC receptor). This receptor is capable of modulating the production of at least one of the serpins, alpha 1-AT. Since the ligand specificity is similar to that previously described for in vivo clearance of serpin-enzyme complexes, the SEC receptor may also be involved in the clearance of certain serpin-enzyme complexes.
Subject(s)
Leukocyte Elastase , Monocytes/metabolism , Pancreatic Elastase/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Serpins/metabolism , Tumor Cells, Cultured/metabolism , alpha 1-Antitrypsin/metabolism , Amino Acid Sequence , Binding, Competitive , Carcinoma, Hepatocellular , Cell Line , Cell Membrane/metabolism , Humans , Kinetics , Liver Neoplasms , Molecular Sequence Data , Peptides/chemical synthesis , alpha 1-Antitrypsin/biosynthesisSubject(s)
Cathepsins/antagonists & inhibitors , Pancreatic Elastase/antagonists & inhibitors , Protease Inhibitors/pharmacology , Amino Acid Sequence , Binding Sites , Cathepsin G , Genetic Variation , Humans , In Vitro Techniques , Kinetics , Leukocytes/enzymology , Pancreatic Elastase/blood , Protease Inhibitors/genetics , Seeds/analysis , Serine EndopeptidasesABSTRACT
Sequence homology comparisons between serum serine protease inhibitors led to the prediction that the C-terminal sequences are functionally equivalent and represent an essential protease binding domain. Inhibition of complement serine protease D cleavage of factor B and of C1s cleavage of C4 by synthetic peptides containing sequences from the C-termini of three serum serine protease inhibitors supports this prediction. These functionally equivalent peptides represent a new class of inhibitors of D and C1s as well as other serum serine proteases.
Subject(s)
Complement Activation , Peptide Fragments/pharmacology , Serine Proteinase Inhibitors , Amino Acid Sequence , Complement C1 Inactivator Proteins , Complement Factor D/antagonists & inhibitors , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
Synthetic peptides based on the amino acid sequence at the site of cleavage of C3 by classical and alternative pathway convertases were found to be poor inhibitors of hemolysis except at concns of 1 mM and higher. Synthetic peptides of a second type, based on the C-terminal sequence of antithrombin III, were more effective; the best among them caused significant inhibition of hemolysis at a concn of 5 microM. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to an antithrombin III sequence was selective, inhibiting the classical pathway with no effect on the alternative pathway at a concentration of 25 microM. Several of the antithrombin III peptides that inhibited hemolysis did not inhibit C4 activation by the classical pathway or activation of C3 by the classical and alternative pathways suggesting that these peptides affect hemolysis by inhibiting enzymes other than C1s and C4b2a of the classical pathway and C3bBb of the alternative pathway.
Subject(s)
Complement C3/biosynthesis , Complement C4/biosynthesis , Hemolysis/drug effects , Peptide Fragments/pharmacology , Serine Proteinase Inhibitors , Amino Acid Sequence , Animals , Antithrombin III/immunology , Complement Activation/drug effects , Complement C3/immunology , Complement C3a , Complement C4a , Molecular Sequence DataABSTRACT
We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.
Subject(s)
Oligopeptides/pharmacology , Pepstatins/pharmacology , Protease Inhibitors , Amino Acids , Amino Alcohols , Animals , Aspartic Acid Endopeptidases , Cathepsin D , Cathepsins/antagonists & inhibitors , Cell Line , Endopeptidases/metabolism , Fibroblasts/metabolism , Humans , Insulin/metabolism , Lung/metabolism , Mice , Pepsin A/antagonists & inhibitors , Protein Conformation , Structure-Activity RelationshipABSTRACT
Prephenate dehydratase from Bacillus subtilis was found to exist in three states of aggregation. A high molecular weight (210,000) species was fully active and the catalytic activity was unaffected by the effectors methionine or phenylalanine. Low concentrations of phenylalanine caused dissociation to a Mr = 55,000 dimer. Heating to 32 degrees C also caused dissociation, but cooling and adding substrate or methionine favored association. When no effectors were present the enzyme eluted from Sephadex columns as a monomer. Both methionine and phenylalanine shifted the equilibrium from the inactive monomer to the active dimeric enzyme. In the presence of a saturating methionine concentration, the dimer possessed the same high activity as did the 210,000-dalton form. Phenylalanine inhibited the dimer, but not the higher molecular weight form. A model involving only three types of sites (catalytic, association-activation, and inhibition) is consistent with the data. It is proposed that phenylalanine is the preferred metabolite for binding both effector sites on the dimer; it binds the association-activation site with higher affinity than the inhibition site, but binding at the latter site has a greater effect on the catalytic rate. Methionine, like phenylalanine, has a hydrophobic side chain but is accommodated only at the association-activation site.
Subject(s)
Bacillus subtilis/enzymology , Hydro-Lyases/metabolism , Methionine/pharmacology , Phenylalanine/pharmacology , Prephenate Dehydratase/metabolism , Allosteric Regulation , Allosteric Site , Kinetics , Macromolecular Substances , Molecular WeightABSTRACT
Synthesis of all four stereoisomers of the novel amino acid statine, 4-amino-3-hydroxy-6-methylheptanoic acid, found in pepstatin, a potent acid protease inhibitor, has been accomplished. Carbobenzoxy-L-valyl-L-valyl-statine tripeptides derived from all four stereoisomers have been prepared and their effect on pepsin activity is compared to that of pepstatin.
Subject(s)
Amino Acids/chemical synthesis , Amino Alcohols/chemical synthesis , Oligopeptides/chemical synthesis , Pepsin A/antagonists & inhibitors , Amino Acids/pharmacology , Amino Alcohols/pharmacology , Hemoglobins/metabolism , Oligopeptides/pharmacology , Pepstatins/pharmacology , StereoisomerismSubject(s)
Lung/drug effects , Oligopeptides/pharmacology , Pepstatins/pharmacology , Proteins/metabolism , Cell Transformation, Viral , Humans , In Vitro Techniques , Lung/enzymology , Lysosomes/drug effects , Lysosomes/enzymology , Protease Inhibitors , Stereoisomerism , Structure-Activity RelationshipSubject(s)
Edible Grain/enzymology , Peptide Hydrolases/analysis , Acids , Biological Availability , Food Handling , WaterABSTRACT
Prephenic acid accumulated in culture filtrates of Neuro-spora crassa has been purified in 66% yield utilizing adsorption chromatography on Sephadex G-10 in the major purification steps.
Subject(s)
Cyclohexanecarboxylic Acids/isolation & purification , Neurospora crassa/analysis , Neurospora/analysis , Barium , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Culture Media , Cyclohexenes , Fractional Precipitation , Mutation , Neurospora crassa/genetics , Pyruvates/isolation & purificationABSTRACT
A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.
Subject(s)
Chymotrypsin , Acetophenones , Amino Acids/analysis , Chemical Phenomena , Chemistry , Chymotrypsin/analysis , Formates , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Protein ConformationABSTRACT
Several known alpha-amino acid analogues and a new compound, N-chloroacetylphosphoramidate, a carbamyl phosphate analogue, were screened as antitumor agents. All gave 50% growth inhibition of cultures of human epidemeroid carcinoma of the nasopharynx at dosage levels of 2-8 mug/ml while showing no activity against L1210 lymphoid leukemia in vivo in BDFi mice.
Subject(s)
Amino Acids/chemical synthesis , Antimetabolites, Antineoplastic/chemical synthesis , Acetamides/chemical synthesis , Acetamides/pharmacology , Acetamides/therapeutic use , Animals , Antimetabolites, Antineoplastic/therapeutic use , Aspartate Carbamoyltransferase/antagonists & inhibitors , Carcinoma, Squamous Cell/drug therapy , Cells, Cultured , Escherichia coli/enzymology , In Vitro Techniques , Leukemia L1210/drug therapy , Methods , Mice , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/pharmacology , Organophosphorus Compounds/therapeutic useSubject(s)
Affinity Labels/chemical synthesis , Amino Acids/metabolism , Proteins/metabolism , Amino Acid Chloromethyl Ketones/chemical synthesis , Amino Acid Chloromethyl Ketones/pharmacology , Bacillus/drug effects , Bacillus/metabolism , Biological Transport, Active , Methods , Neurospora/drug effects , Neurospora/metabolismABSTRACT
The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.