Donald T. Elmore

Isolation of glucagon-like immunoreactivity of gut by affinity chromatography on anti-glucagon antibodies coupled to sepharose 4B

Abstract 1. 1. Antibodies raised in rabbits against porcine pancreatic glucagon were coupled to Sepharose 4B. The resultant complex was used to purify porcine pancreatic glucagon and glucagon-like immunoreactivity of pig ileum. The purified glucagon-like immunoreactivity was eluted from gel-filtration columns at positions corresponding to proteins of mol. wt. 12 000 (large glucagon-like immunoreactivity) and 3500 (small glucagon-like immunoreactivity). The former fraction accounted for over 95% of the total immunoreactivity. 2. 2. Large glucagon-like immunoreactivity contained at least three N-terminal amino acids, glycine, alanine and methionine, and was separated into three immunoreactive components by electrophoresis. 3. 3. Large glucagon-like immunoreactivity did not display the biological activity of pancreatic glucagon.

Enzymic iodination of the histidyl residue of secretin: a radioimmunoassay of the hormone.

Abstract Lactoperoxidase (EC 1.11.1.7) was a more efficient catalyst than chloramine-T for the incorporation of 125 I into secretin. Satisfactory radioimmunoassays were performed using the labelled product. Total hydrolysis and Edman degradation of the iodinated secretin showed that only the N-terminal histidyl residue of the hormone had been iodinated. Poly( l -histidine) was also iodinated using lactoperoxidase.

Enzymic iodination of polypeptide hormones for radioimmunoassay

Abstract The use of lactoperoxidase (EC 1.11.1.7) and chloramine-T as reagents for the incorporation of 125 I into polypeptide hormones (glucagon, gastrin, insulin, B-chain of insulin) for radioimmunoassay, was compared. In general, results were more reproducible when the enzyme was used; 70–80% of the label present was incorporated into the polypeptides and the iodinated hormones had higher affinity for antibody (over 80% was bound by excess antibody) resulting in better standard curves in radioimmunoassay. The enzymic method appears to give rise to fewer side-reactions.

Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand.

The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed.

THE DEGRADATION OF BOVINE AND HUMAN PROTHROMBIN BY HUMAN POLYMORPHONUCLEAR LEUKOCYTE CATHEPSIN G

Cathepsin G, isolated from human polymorphonuclear leukocytes, was found to effect rapid and specific degradation and biological inactivation of bovine and human prothrombin in the absence of calcium ions with the formation of two peptide fragments from the N-terminal end of the molecule. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular weights of the fragments were 5,000 and 17,500. Proteolysis of prothrombin by cathepsin G was inhibited by calcium ions. Leukocyte proteinases such as cathepsin G may be responsible for haemorrhagic disorders associated with myelocytic leukaemia and septicaemia.

The hydrolysis of chemically modified proteins by factor Xa and thrombin

Bovine thrombin and Factor Xa were shown to hydrolyse slowly several chemically modified proteins. Both enzymes hydrolyse the proteins at trypsin-susceptible bonds, with arginine, lysine or the synthetically generated S-(beta-aminoethyl)cysteine at the P1 position. Both enzymes, however, cleave at far fewer sites than trypsin. The presence of highly polar groups in the P2 position appears to hinder hydrolysis by Factor Xa or thrombin. The presence of hydrophobic or neutral amino acids around this site may make the site more susceptible to hydrolysis. Differences in the hydrolysis patterns between thrombin and Factor Xa are observed.

An enzyme-scissile linker for solid-phase peptide synthesis

We describe a new linker that contains a phosphodiester moiety for solid-phase peptide synthesis; after completion of the cycle of coupling and deprotection steps, the phosphodiester can be cleaved with phosphodiesterase.

The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.

The esterolytic specificity of bovine thrombin and factor Xa

Steady state kinetics are compared for the hydrolysis of t-butoxycarbonyl-L-lysine methyl ester and several peptidyl lysine methyl esters catalysed by bovine thrombin and Factor Xa. Thrombin-catalysed reactions have lower Km values and higher kcat/Km values than do reactions catalysed by Factor Xa. Values of kcat are comparable and do not show any particular trend. The best substrate in the present series was t-butoxycarbonylglycylglycyl-L-lysine methyl ester. Thrombin and Factor Xa may possess a hydrophobic region near the P2 binding site which is unfavourable for either asparagine or D-alanine but which readily accommodates glycine, L-alanine or L-phenylalanine. The major improvement in Factor Xa hydrolysis occurred with the occupation of the P2 site by an amino residue while for thrombin the major improvement occurred with the occupation of the P3 site.

Azasulfonamidopeptides as peptide bond hydrolysis transition state analogues. Part 2. Potential HIV-1 proteinase inhibitor

The synthesis of [N-benzyl-N′-(Nα-benzyloxycarbonyl-L-asparaginyl)hydrazino]sulfonyl-L-prolyl-L-isoleucyl-L-valine methyl ester 4, a potential HIV-1 proteinase inhibitor, is described.