John M. Prescott

[44] Aeromonas aminopeptidase

Publisher Summary Aeromonas aminopeptidase is a heat-stable extracellular enzyme that can be isolated in high yield from culture filtrates of the marine bacterial species, Aeromonas proteolytical. It is readily prepared in physically homogeneous form, free from traces of endopeptidase activity. The production and isolation of this aminopeptidase are described in this chapter. It details the procedure for enzyme production and enzyme isolation. The key properties of the enzymes, such as the purity, physical properties, stability, specificity and catalytic properties, inactivation and Inhibitions are described. The Leucyl-β -naphthylamide Assay, Leucyl-p-nitroanilide Assay, Gelatin-Digesting Test for Endopeptidase and the Oligopeptide Test for Endopeptidase Activity are also discussed in detail.

Aeromonas aminopeptidase: Purification and some general properties

Abstract An aminopeptidase was purified 87-fold from culture filtrates of the marine bacterium Aeromonas proteolytica . The stability of the aminopeptidase at 70 ° permitted the use of a heat treatment to inactivate a proteinase from which the aminopeptidase was otherwise difficult to separate. Purification of the aminopeptidase from heat-treated culture filtrate was accomplished by chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. Moving-boundary electrophoresis, ultracentrifugation, and substrate specificity studies indicated a high degree of homogeneity for the preparations. The enzyme hydrolyzed a number of peptides, amino acid amides, and amino acid β-naphthylamides. It was specific for substrates having a free α-amino group on a residue of the l -configuration; the nature of the amino acid in the N-terminus exerted a considerable effect on hydrolytic rates. The aminopeptidase, as isolated, did not require the addition of activating ions, but was strongly inhibited by ethylenediaminetetraacetate (EDTA). Addition of Zn ++ or Co ++ to the EDTA-inhibited enzyme restored activity to the original level; Mn ++ was partially effective, and Mg ++ , Ca ++ , and Ni ++ were ineffective. Analysis of several individual preparations revealed the presence of zinc in the aminopeptidase. A molecular weight of 29,500 was calculated from measurements of sedimentation velocity and diffusion.

Leucostoma peptidase A: a metalloprotease from snake venom.

Abstract The substrate specificity and metal ion content of leucostoma peptidase A from the venom of Agkistrodon piscivorus leucostoma were investigated. The enzyme was found to contain calcium and zinc in a 2:1 ratio, and on the basis of a mol. wt of 22 500, it appeared that these values represented 2 gatoms of calcium and 1 gatom of zinc per mole. Activity was inhibited by exposure of the enzyme to o-phenanthroline in the presence of excess calcium, but the activity of enzyme treated in this manner was restored by the addition of zinc ions. Exposure to EDTA, however, removed both zinc and calcium and resulted in irreversible inactivation. Leucostoma peptidase A hydrolyzed several N-substituted or doubly substituded dipeptides in which an aromatic or a basic residue contributed the amino group. It hydrolyzed four of nine polyamino acids tested, and cleaved the B-chain of oxidized insulin at positions Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Gly20-Glu21, Gly23-Phe24, and Phe24-Phe25. These data indicate that the properties of leucostoma peptidase A are those of the general class known as neutral proteases, examples of which have previously been reported only in microorganisms.

Isolation and properties of an aminopeptidase from Bacillus licheniformis

Abstract An extracellular aminopeptidase, purified 465-fold from culture filtrates of Bacillus licheniformis , was found to be a metalloenzyme consisting of a single peptide chain. Sedimentation equilibrium yielded a molecular weight of 43,270 and two polyacrylamide electrophoretic procedures gave values of 37,500 and 36,000, respectively. The activity of the enzyme was inhibited severely by 1,10-phenanthroline and to a lesser extent by EDTA, cyanide, and fluoride. The addition of Co 2+ ions greatly stimulated enzymatic activity, but analysis of the purified enzyme revealed the presence of zinc, not cobalt, in stoichiometric quantities. Moreover, the ratio of zinc to protein was found to increase during fractionation, reaching a final value corresponding to 1 g-atom/mol. The aminopeptidase possessed characteristics of a euglobulin, sparingly soluble in water and dilute buffer solutions, but soluble in buffers containing higher concentrations of salts. Both activity and pH optimum were substantially influenced by ionic strength; as the latter was increased over the range from 0.01 to 0.1, activity increased and the pH optimum was shifted to more acidic values. Enzymatic activity was affected by the identity of the buffer, being markedly greater in Tris-HCl than in sodium barbital and strongly inhibited by phosphate. The Bacillus aminopeptidase hydrolyzed substrates with unsubstituted amino groups of the l configuration, including dipeptides, aminoacylnaphthylamides, and amino acid amides.

Anion exchange chromatography of oxidized insulin peptides.

Abstract The A and B chains of performic acid-oxidized bovine insulin were separated by a rapid, simple procedure involving chromatography on DEAE-Sephadex. Elution was accomplished with a gradient of ammonium formate, which was removed from the final products by sublimation to yield salt-free preparations of the two peptides. The elution gradient produced good resolution of the A and B chains, which were collected in sharp fractions. The B chain emerged in a high state of purity, as judged by quantitative amino acid analysis. The A chain frequently contained traces of contamination which could be removed by rechromatography on the DEAE-Sephadex.

Kinetics of inhibition of Aeromonas aminopeptidase by leucine methyl ketone derivatives

Abstract A number of methyl ketones have been prepared from l -leucine and found to be competitive inhibitors of Aeromonas aminopeptidase. These inhibitors were leucine methyl ketone (K i 18 μ m ), leucine chloromethyl ketone (K i 0.67 μ m ), and leucine bromomethyl ketone (K i 0.20 μ m ), and the corresponding succinimido derivative (K i 170 μ m ), succinamic acid derivative (K i 6.9 μ m ) and phthalimido derivative (K i 140 μ m ). Reversible inhibition was observed for all of the inhibitors tested, indicating that the active site of this enzyme is not alkylated or acylated by the nucleophile-sensitive components of some of the inhibitors. The chloromethyl ketones derived from l -leucine and l -phenylalanine were found to have the same relative binding constants as the substrates, l -leucinamide and l -phenylalaninamide.

Isolation and Some Characteristics of Glutathione Reductase from Rabbit Erythrocytes

Summary Glutathione reductase from rabbit erythrocytes was purified to homogeneity and found to be a monomer with a mol wt of 60,000. Both NADPH and NADH were capable of acting as cofactors for the reduction of GSSG and the following kinetic values were obtained: K m, gssg =120 μM; K m, nadph = 37 μM; V max = 23 μmoles NADPH/min/mg protein, K m, nadh = 420 μM; V max = 3 jumoles NADH/min/mg protein. Rabbit erythrocyte GR exhibited substrate inhibition, and was susceptible to inhibition by p-hydroxymercuribenzoate under certain conditions. The invaluable assistance of Mrs. Mary E. Bayliss and Mrs. Stella H. Wilkes with some of the experiments is gratefully acknowledged.

A comparative study of proteolytic activities in the venoms of some North American snakes

Abstract 1. 1. Venoms from four species of North American snakes were investigated with respect to their proteolytic activities. All of the venoms readily hydrolyzed protein substrates and certain esters. 2. 2. The esterase and proteinase activities differed in pH optima and in their susceptibility to inhibition by diisopropylphorofluoridate and by phenylmethanesulfonyl fluoride. 3. 3. The venoms possessed no carboxypeptidase or true aminopeptidase activity, but measurable amounts of arylamidase activity were present in all of the venoms. 4. 4. Proteolysis by the venom from Agkistrodon piscivorus piscivorus was inhibited by reducing agents and chelating agents; inhibition by the latter could not be reversed by alterations in pH or magnesium ion concentration. 5. 5. The effects of several divalent ions on proteolysis by the venoms were tested. None of these ions greatly enhanced protein hydrolysis, but several were inhibitory.

An aminotransferase specific for the d-enantiomorph of methionine

Abstract An enzyme was isolated from germinating peanut seed and shown to be an aminotransferase specific for the d -enantiomorph of methionine. The keto acid of methionine, α-keto-γ-methylthiobutyrate, was isolated from the reaction mixture and identified. Of the keto acids tested pyruvic acid was the most effective acceptor for the amino group of methionine. A small amount of enzyme was isolated which gave only one band on disc gel electrophoresis.

Multiple proteolytic enzymes of Bacillus licheniformis

Abstract By the use of synthetic substrates, organofluoride inhibitors, and heat denaturation, three types of proteolytic enzymes were identified in culture filtrates of Bacillus licheniformis . The proteinases detected were (i) an enzyme with high activity toward N -acetyl- l -tyrosine ethyl ester (ATEE) and toward proteins, highly susceptible to organofluoride inhibition, and rapidly denatured at 65 °C; (ii) a proteinase with low activity toward ATEE, slowly inhibited by organofluorides, and inactivated relatively slowly at 65 °C; and (iii) an aminopeptidase, unaffected by organofluorides, and stable at 65 °C for 60 minutes. In the unfractionated enzyme system, esterolytic activity was greatest toward N -substituted esters of aromatic l -amino acids, but substituted esters of the d -enantiomorphs were not hydrolyzed. Unsubstituted esters of l -amino acids were not susceptible, and N -substituted esters of basic amino acids were hydrolyzed only slowly, as were the amides of N -blocked aromatic amino acids. Gel-infiltration on Sephadex G-200 separated two fractions which varied greatly in their relative activities toward ATEE and N -benzoyl- l -arginine ethyl ester (BAEE), thus furnishing further evidence for the presence of two endopeptidases. Gross proteolysis of the unfractionated system was enhanced by Ca ++ ions, and was inhibited by EDTA. The aminopeptidase was stimulated by Co ++ ions.