Tsau-Yen Lin

Methyl-14C-glycinated hemoglobin as a substrate for proteases

Abstract A simple radioisotopic assay for the proteolytic enzyme activity with methyl- 14 C-glycinated bovine hemoglobin as the substrate has been developed. The radioactive hemoglobin was prepared by the reaction of hemoglobin with a water-soluble carbodiimide and methyl [ 14 C]glycinate in 5 M guanidine hydrochloride.

Phosphinic acid inhibitors of matrix metalloproteinases

Abstract The matrix metalloproteinase stromelysin-1 (MMP-3) is inhibited more strongly by peptidyl phosphinic acid 7 than by its corresponding phosphonamidate and phosphonate analogs. Extending a benzyl group at P′ 1 to a phenylethyl group in 8 further increases the potency (K i = 1.4 nM). Enhanced potency with an extended substituent into the P 3 region was observed.

Inhibition of stromelysin-1 (MMP-3) by peptidyl phosphinic acids

Abstract A series of phosphinic acid-containing peptide inhibitors of human stromelysin-1 (MMP-3) were prepared. The P1 through P3 subsites were varied in a systematic manner on analogs possessing an invariant P1′-P3′ segment. The in vitro activity of these compounds as inhibitors of stromelysin and collagenase is discussed.

Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, that is human renin specific and is resistant to cathepsin D

A synthetic tetradecapeptide, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, which corresponds to the 13 amino terminal residues of human angiotensinogen plus a carboxy terminal serine to replace a suggested site of carbohydrate attachment, has been shown to be a good substrate for human kidney renin. At pH 7.2 and 37 degrees C the KM or Michaelis constant was 8.4 +/- 2.9 microM, and the VM or velocity at infinite tetradecapeptide concentration was 11.3 +/- 2.4 mumol angiotensin I made per hour per milligram renin. The tetradecapeptide was highly resistant to cleavage by mouse submaxillary renin. The tetradecapeptide was also slowly cleaved by human liver cathepsin D, by rabbit lung angiotensin-converting enzyme, and by reconstituted human serum, but did not yield angiotensin I. Thus, this synthetic renin substrate should permit more specific measurement of human kidney renin activity.

Quantitation of immune complexes by competitive inhibition of binding of Clq to insoluble IgG aggregates

An assay system for the binding of Clq to insoluble IgG aggregates was found useful for the quantitation of immune complexes in biological fluids. The assay, both easily and rapidly performed, is based on the competition of Clq binding substances with IgG aggregates. Serum from rheumatoid arthritis patients showed an increase in Clq binding substances over normal serum and this increase could be abolished by pretreatment of the serum with D-penicillamine.

Incorporation of l-leucine and d-glucosamine into skin fibroblasts derived from cystic fibrosis and normal individuals

Abstract Cultured skin fibroblasts from patients with diagnosed cystic fibrosis were compared with those from normal individuals in respect to incorporation of l -leucine or d -glucosamine into the subcellular components, to uptake of α-isobutyric acid and calcium, and to (Na+-K+)ATPase activity, etc. No significant difference which could be attributed to the disease-specific basic metabolic abnormality was demonstrated. Cystic fibrosis skin fibroblasts may not express the primary genetic defect at the level of generalized transport mechanism and overall protein metabolism.

Human polymorphonuclear leukocyte collagenase and gelatinase: Comparison of certain enzymatic properties

Collagenase and gelatinase of human PMN leukocytes were separated by serial chromatography. The enzymes were shown to be similar in latency, activatability, chromatographic and electrophoretic behavior and the response to inhibitors. They recognize the same peptide linkage for cleavage, only each with a distinct difference in the effect caused by the secondary binding sites of the substrate molecules.

Limited Degradation of Gelatin Substrate by Cathepsin D

Cathepsin D has been demonstrated to degrade the protein backbone of proteinpolysaccharide. In this study we present evidence that cathepsin D has only a limited yet specific capacity to degrade gelatin. Similar to the effects of pepsin on collagen, cathepsin D degraded β chains of pure gelatin to α chains but showed no capacity to cleave at a site other than the N-terminal cross-link region. These data suggest that it is unlikely that cathepsin D has a major role in any phase of collagenolysis.

Effects of human polymorphonuclear leukocyte collagenase on sub-component C1q of the first component of human complement.

Abstract The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase. The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA. Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.

Inhibition of the local hemorrhagic Shwartzman reaction by an acid proteinase inhibitor, pepstatin

Nachweis, dass das lokaleShwartzman-Phänomen in Kaninchen durch intradermale oder i.v. Gabe von Pepstatin, einem sauren Proteinase-Hemmer, oder von Sojabohnen-Trypsin-Hemmer kurz vor der auslösenden Injektion von bakteriellem Endotoxin unterdrückt werden kann.