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J Travis

Publications and source records attributed to J Travis.

At least 253 records · Page 14Linked to original sources
PubMed1986

Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases.

The rates of interaction of a number of serine proteinases with a mutant form of alpha 1-proteinase inhibitor (referred to as alpha 1-proteinase inhibitor (Pittsburgh)), in which a methionine-358 to arginine-358 mutation has occurred, have been determined. An approximately 6,000-fold increase in the second order association rate constant with human thrombin was observed (48 M-1 X s-1 for the normal protein to 3.1 X 10(5) M-1 X s-1 for the arginine mutant), confirming previously observed data using bovine thrombin (Owen, M.C., Brennan, S.O., Lewis, J.H. & Carrell, R.W. (1983) New England J. Med. 309, 694-698). However, substantial increases in the rates of association with other trypsin-like enzymes were also noted, indicating that the replacement of methionine by a basic residue affects all serine proteinases with this kind of specificity. There was a marked decrease in the rates of interaction of the Pittsburgh mutant with both human neutrophil elastase and porcine pancreatic elastase, the inhibitor being converted into lower molecular mass fragments after interaction with either enzyme. Butanedione caused a substantial loss in the inhibitory activity of the arginine mutant, while having no effect on the normal protein. These data, when compared to those previously reported for differences in reaction rates between normal and oxidized alpha 1-proteinase inhibitor (Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, 3931-3934), are consistent with the interpretation that the amino acid in the P1-position at the reactive site of this protein has a marked effect on determining its primary specificity.

Blood Proteins↗
PubMed1986

Target enzymes for plasma proteinase inhibitors.

The control of proteolytic activity in tissues is primarily under the influence of plasma inhibitors which function to rapidly inactivate specific target proteinases by either covalent interactions or trapping reactions. Each inhibitor is designed to control a specific proteolytic event, although many may show weaker activities against other proteinases. Kinetic experiments, however, indicate that reactions with target enzymes are much more rapid than with other and that modification of the inhibitor dramatically interferes with the rate at which inhibition occurs. This latter effect is almost certainly due to the fact that the inhibitor itself acts as a perfect substrate for the proteinase in question, resulting in a rapid association with the enzyme followed by a very slow dissociation so that the enzyme is essentially trapped in a complex.

Binding Sites↗
PubMed1985

Isolation and properties of recombinant DNA produced variants of human alpha 1-proteinase inhibitor.

Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human alpha 1-proteinase inhibitor, representing 10% of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated with one being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, and both had shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasma alpha 1-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing protein was readily inactivated. The specificity of alpha 1-proteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or pancreatic elastase, "trypsin-like" serine proteinases not being inactivated at all. These data indicate the potential importance of mutant forms of proteinase inhibitors, produced by recombinant DNA technology, as therapeutic agents for the inactivation of excess proteinases of a specific type in tissues.

Blood Proteins↗
PubMed1985

Differential effects of oxidizing agents on human plasma alpha 1-proteinase inhibitor and human neutrophil myeloperoxidase.

Human alpha 1-proteinase inhibitor is easily susceptible to inactivation because of the presence of a methionyl residue at its reactive site. Thus, oxidizing species derived from the myeloperoxidase system (enzyme, H2O2, and C1-), as well as hypochlorous acid, can inactivate this inhibitor, although H2O2 alone has no effect. Butylated hydroxytoluene, a radical scavenger, partially protects alpha 1-proteinase inhibitor from the myeloperoxidase system and completely protects it from hypochlorous acid. Each oxidant also reacts differently with the inhibitor, in that the myeloperoxidase system and hypochlorous acid can each oxidize as many as six methionyl residues, but hypochlorous acid can also oxidize a single tyrosine residue. Myeloperoxidase can be inactivated by hypochlorous acid, by autoxidation in the presence of H2O2 and C1-, as well as by H2O2 alone. Butylated hydroxytoluene completely protects this enzyme from hypochlorous acid inactivation, does not affect the action of H2O2, and enhances autoinactivation. As many as six methionyl residues and two tyrosine residues could be oxidized during autoxidation and six methionine residues by H2O2 alone. Eight methionine residues and one tyrosine residue could be oxidized by hypochlorous acid. The tyrosine residue in myeloperoxidase was oxidized only at a relatively high concentration (600 microM) of hypochlorous acid at which point the enzyme simultaneously and completely lost its enzymatic activity. Loss of activity of myeloperoxidase could also be correlated with the loss of the heme groups present in the enzyme when a relatively high concentration of hypochlorous acid (600 microM) was used and also during autoxidation. It appears that once there is sufficient oxidant to modify one of the tyrosine residues, the heme group itself becomes susceptible.

Amino Acids↗
PubMed1985

Human leukocyte cathepsin G. Subsite mapping with 4-nitroanilides, chemical modification, and effect of possible cofactors.

The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.

Anilides↗
PubMed1985

Inactivation of bradykinin and kallidin by cathepsin G and mast cell chymase.

Human neutrophil cathepsin G and human skin chymase can inactivate bradykinin by cleavage at the carboxy terminal phenylalanyl-arginyl peptide bond of this polypeptide. The mast cell enzyme is far more effective than cathepsin G, the rates of hydrolysis being comparable to that found for angiotensin I to angiotensin II conversion (C.F. Reilly, D. Tewksbury, N. Schechter, and J. Travis, J. Biological Chemistry 257:8619-8622). This ability to both inactivate bradykinin and accelerate the production of angiotensin II may be of significance in the development of biochemical events associated with inflammation.

Amino Acid Sequence↗
PubMed1985

Primary structure of the reactive site of human C1-inhibitor.

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.

Amino Acid Sequence↗
PubMed1985

The reactive site of human inter-alpha-trypsin inhibitor is in the amino-terminal half of the protein.

Human inter-alpha-trypsin inhibitor has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G. The protein was cleaved into two major fragments without loss of activity by incubation with Serratia marcescens metalloproteinase, and these were separated by ion-exchange chromatography. Inhibitory activity was found in only one of the fragments, the amino-terminal sequence of which was found to be identical with that of the native protein, as well as with that reported earlier for the urinary trypsin inhibitor. It may thus be concluded that the reactive site of the inter-alpha-trypsin inhibitor is located in the amino-terminal region.

Amino Acid Sequence↗
PubMed1984

A genetically engineered mutant of alpha 1-antitrypsin protects connective tissue from neutrophil damage and may be useful in lung disease.

The effectiveness of a genetically engineered mutant of human alpha 1-antitrypsin (358 Met----Val) as an inhibitor of connective tissue breakdown was tested in a model of inflammation. The degradation of basement membrane collagen by stimulated neutrophils was efficiently inhibited by a tenfold lower concentration (0.2 mg/ml) of the mutant inhibitor than of the normal alpha 1-antitrypsin (2.4 mg/ml). Effective inhibition by normal alpha 1-antitrypsin occurred at much lower concentrations when azide or catalase was added, or when normal neutrophils were replaced by those from a donor with chronic granulomatous disease. These results confirm that neutrophils augment tissue proteolysis by the oxidative inactivation of the methionine at the reactive centre of alpha 1-antitrypsin. The replacement of this methionine by valine gives an effective inhibitor that is not inactivated by neutrophil oxidants. The availability of this genetically engineered mutant suggests the possibility of prophylaxis of lung dysplasias, notably emphysema, and of the shock syndromes associated with massive neutrophil activation.

Basement Membrane↗
PubMed1984

Structural and functional characterization of the abnormal Z alpha 1-antitrypsin isolated from human liver.

alpha 1-Antitrypsin has been isolated from liver inclusion bodies of a subject with a homozygous Z deficiency. The inhibitor was recovered in a fully active form by extraction in high salt at either pH 2.0 or pH 8.0. Carbohydrate analysis indicated a protein in the 'high mannose' form, and this was collaborated by its sensitivity to endo-beta N-glucosaminidase. These data suggest that the abnormal alpha 1-antitrypsin is blocked in the secretory pathway prior to its entrance into the Golgi, and that this blockage is not due to a gross misfolding of the polypeptide.

Carbohydrates↗
PubMed1984

Kinetics of association of human proteinases with human alpha 2-macroglobulin.

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.

Binding Sites↗
PubMed1984

Effect of neutrophil cathepsin G on elastin degradation by neutrophil elastase.

Human neutrophil cathepsin G was found to be unable to significantly stimulate the degradation of either bovine or human elastin by neutrophil elastase, using four different procedures to monitor digestion. A range of stimulations from 1.1 to 2.9-fold was found, with a 2.0-fold stimulation being the average found with the assays tested. These results contrast with those reported by Boudier et al. [(1981) J. Biol. Chem. 256, 10256-10258] who reported a five- to seven-fold stimulation of elastolysis of human lung elastin by cathepsin G, when present at a 2:1 molar ratio relative to elastase. Significantly, we found little stimulation of elastolysis with either human or bovine lung elastin as substrate while Boudier et al. found stimulation only with the human elastin. Thus, it would appear that cathepsin G does not play a predominant role as an elastolytic enzyme; rather, its role in this case may be one of binding to non-productive sites on the elastin surface.

Animals↗
PubMed1984

Kinetic and chemical evidence for the inability of oxidized alpha 1-proteinase inhibitor to protect lung elastin from elastolytic degradation.

The oxidation of human alpha 1-proteinase inhibitor results in the conversion of this protein into a form which cannot protect lung elastin from degradation by elastolytic proteinases. Data indicate that this is primarily because of the lowering of the association rate between the modified inhibitor and neutrophil elastase, as well as in a change in Ki from near 10(-14) to near 10(-10)M. This is consistent with the hypothesis that oxidation of alpha 1-proteinase inhibitor in the lung by cigarette smoke results in a lowering of the protection of this organ from elastolytic degradation.

Blood Proteins↗
PubMed1983

Amino acid sequence at the reactive site of human alpha 1-antichymotrypsin.

The reactive site of human alpha 1-antichymotrypsin has been identified as encompassing a leucyl-seryl bond at the apparent P1 and P'1 positions. This has been determined by dissociation of complexes of the inhibitor with bovine alpha-chymotrypsin, followed by identification of new NH2-terminal sequences, as well as by proteolytic inactivation by porcine pancreatic elastase. The latter results in peptide bond cleavage between the apparent P5 and P4 positions of the inhibitor, yielding a fragment whose sequence overlaps with that obtained through complex dissociation. Some homology with the sequence obtained and that already reported for both antithrombin III and alpha 1-proteinase inhibitor can be noted.

Amino Acid Sequence↗
PubMed1983

Structural alterations in alpha 1-antichymotrypsin from normal and acute phase human plasma.

Human alpha 1-antichymotrypsin, isolated at pH 8.0 from both normal and acute phase plasma, has been found to have two different amino terminal sequences despite the fact that inhibitory activities are unchanged. In normal plasma over 90% of the protein has an amino terminal sequence beginning with aspartic acid and less than 10% with arginine. However, in acute rheumatoid arthritis plasma 55% of the inhibitor begins with arginine and the remainder with aspartic acid. Sequence studies indicate that a fifteen amino acid peptide fragment has been cleaved to yield the arginine protein. Human alpha 1-proteinase inhibitor also shows this heterogeneity, but the ratios do not change between normal and acute phase plasma. It may well be that the missing peptide has some biological activity manifested only in the acute phase state.

Acute Disease↗