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Biomedical subjects

A Light

Publications and source records attributed to A Light.

At least 55 records · Page 3Linked to original sources
PubMed1979

Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.

Animals↗
PubMed1979

The preparation and properties of bovine enterokinase.

Bovine enterokinase was purified from duodenal mucosa. The purification included an initial extraction with 2% deoxycholate, ammonium sulfate fractionations, DEAE-cellulose chromatography, and affinity chromatography on basic pancreatic trypsin inhibitor (Kunitz) (PTI)-Sepharose. The purified enzyme contained 35% carbohydrate; it had a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Enterokinase hydrolyzed lysine and arginine substrates and slowly reacted with the trypsin active site titrant 4-methylumbelliferyl-p-guanidinobenzoate. The enzyme activated bovine trypsinogen with kinetic parameters similar to those of other preparations of enterokinase. Bovine enterokinase was inhibited by Kunitz pancreatic trypsin inhibitor with a Kassoc of 2 X 10(8) M-1 and only weakly by other proteinase inhibitors. The amino acid composition differed from bovine enterokinase isolated from duodenal contents (Anderson, L.E., Walsh, K.A., and Neurath, H. (1977) Biochemistry 16, 3354-3360). The mucosal enzyme and the duodenal contents enzymes also differed in the size of the heavy and light chains. The mucosal enterokinase more closely resembled the properties of porcine enterokinase (Baratti, J., Maroux, S., Louvard, D., and Desnuelle, P. (1973) Biochim. Biophys. Acta 315, 147-161). The amino acid composition and size of the light chain were also similar to bovine trypsin.

Amino Acids↗
PubMed1979

Solid-phase active site inhibitors of proteinases.

Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid-phase active site directed inhibitor of chymotrypsin-like proteolytic enzymes. The solid-phase reagent inhibited 20 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose-bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin-like enzymes. Sepharose-MethionylLysyl chloromethyl ketone inactivated and bound about 6.8 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active-site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid-phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active-site histidine.

Animals↗
PubMed1976

Disulfide bond-modified trypsinogen. Role of disulfide 179-203 on the specificity characteristics of bovine trypsin toward synthetic substrates.

Disulfide 179-203 of trypsinogen was cleaved and the free sulfhydryls were modified by S-carboxymethylation, S-carboxyamidomethylation, or by S-aminoethylation. The enzymatic properties of the activated, modified zymogens toward specific and nonspecific trypsin substrates were studies. The three S-alkylated trypsins hydrolyzed N-benzoylarginine ethyl ester and N-tosyl-lysine methyl ester with Kcat values similar to those of trypsin but with Km values that were increased by 2 to 3 orders of magnitude. The binding constant of the competitive inhibitor benzamidine to the S-alkylated trypsins was increased by 2 orders of magnitude by the modifications. The association constant of soybean trypsin inhibitor with S-carboxyamidomethyl trypsin was several orders of magnitude less than normal. Hydrolysis of benzoyl arginine amide with S-carboxymethyl trypsin was not detected, but this was ascribed to poor binding since a KI of 0.3 M was estimated from competitive inhibition studies. The altered kinetics did not depend on the type of chemical group used for the S-alkylated derivative, nor were the kinetics of any derivative significantly influenced by changes of pH or ionic strength. Nonspecific substrates, such as acetylglycine p-nitrophenyl ester and p-nitrophenylacetate, were hydrolyzed at equal rates by both trypsin and disulfide-modified trypsins, and both substrates had identical kcat/Km ratios with unmodified trypsins. For S-alkylated trypsins, kcat/km with lysine and arginine substrates were lower than normal and were the same order of magnitude as the values found for nonspecific substrates, suggesting normal catalytic behavior but a loss of specificity in binding substrates. The kinetic evidence suggested that the role of disulfide 179-203 in the mechanism of action of trypsin is to maintain the geometry of the specificity pocket by keeping appropriate residues of the pocket in a rigid framework.

Animals↗
PubMed1975

Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.

The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.

Animals↗