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Purification and characterization of a post-proline dipeptidyl aminopeptidase from Streptococcus cremoris AM2

The present study describes the purification of a post-proline dipeptidyl aminopeptidase from the cytoplasm of Streptococcus cremoris AM2. On the basis of its elution from a calibrated Sephadex G200 column, the enzyme had a molecular weight of 117000 and exhibited a broad pH optimum activity between 6·0 and 9·0. The activity was most comprehensively inhibited by phenylmethylsulphonylfluoride and more modestly inhibited by 1,10-phenanthroline and 8-hydroxyquinoline but not by EDTA. A range of peptides containing either proline or alanine as the penultimate amino acid residue could act as substrates. The presence of proline on the carboxy side of the scissile bond prevented hydrolysis. However the enzyme could release Pro-Pro from Pro-Pro-Gly-Phe-Ser-Pro. The significance of this substrate specificity is considered in the context of removal of either single proline residues or prolylproline sequences from oligopeptides during cheese ripening.

Prolidase activity of Lactococcus lactis subsp. cremoris AM2: partial purification and characterization

Prolidase activity from cytoplasm of Lactococcus lactis subsp. cremoris (Streptococcus cremoris) AM2 was partially purified. The enzyme had M r 42000 and optimum activity between pH 7·35 and 8·25 in citrate, phosphate and borate buffers while in a universal buffer system an optimum pH between 8·3 and 9·0 was observed. The activity was strongly inhibited by the chelating agents E.DTA, 1,10-phenanthroline and 8-hydroxyquinoline. Inhibition was also noted with dithio-threitol, N -ethylmaleimide and bacitracin. The enzyme was active on all amino-acylproline substrates tested except Gly-Pro and Gip-Pro and also showed activity against Pro-Pro. While most prolyl amino acids tested were not hydrolysed, hydrolysis was noted with Pro-Ala and Pro-Val. K m values of 20 mM and 10 mM were obtained with Phe-Pro and Met-Pro respectively; however, substrate inhibition was observed with Ile-Pro and Leu-Pro.

Purification and characterisation of an aminotripeptidase from cytoplasm of Lactococcus lactis subsp. cremoris AM2

Abstract An aminopeptidase was purified 120-fold with a 78% recovery from the cytoplasm of Lactococcus lactis subsp. cremoris AM2. The purified enzyme exhibited no activity on dipeptides, dipeptideamides, tripeptideamides or tetrapeptides. As activity was observed only with tripeptides, from which it released the N -terminal amino acid, the enzyme was adjudged to be a strict aminotripeptidase. The enzyme had a Mr of 105 000 and showed one band, corresponding to an Mr of 55 000 on SDS-PAGE electrophoresis. Inhibition by EDTA. 8-hydroxyquinoline and 1.10 phenanthroline indicated that the peptidase was a metallo enzyme. Dithiothreitol, bestatin and amastatin caused total inhibition, whereas p -chloromercuribenzoate, bacitracin and phenyl methyl sulphonyl fluoride were without effect. K m values for tripeptides were within the range 0·18 (Leu-Leu-Leu) to 0·38 mM (Trp-Gly-Gly). Substrate inhibition was noted with some tripeptides at concentrations above 1·5 mM.

Effects of allopurinol and of oxypurinol on turkey liver xanthine dehydrogenase.

Allopurinol a potent inhibitor of xanthine:oxygen oxidoreductase (EC 1.2.3.2) activity of milk xanthine oxidase is converted to oxypurinol by the enzyme [l] . Enzyme incubated with allopurinol rapidly loses the ability to catalyse the oxidation of xanthine by 02. By contrast preliminary incubation with oxypurinol causes no decrease in activity unless xanthine is also present [2]. Inactivation results from complex formation between oxypurinol and the enzyme-bound molybdenum in the tetravalent state [3,4]. The effects of allopurinol and oxypurinol on the xanthine dehydrogenases (purine hydroxylases) from Aspergillus nidulans differ considerably from those on xanthine oxidase (Scazzocchio, personal communication; Lewis, Hurt, Sealy-Lewis and Scazzacchio, in preparation). Work with crude extracts showed both agents to be pseudo-irreversiile inhibitors of purine hydroxylase I. However, inactivation by allopurinol appears not to be due to its conversion to oxypurinol. With purine hydroxylase II, allopurinol is a competitive inhibitor while oxypurinol shows anticompetitive inhibition. In this case neither compound forms an inactive complex with the enzyme. ln order to determine whether these findings are unique to the enzymes from Aspergillus or shared by xanthine dehydrogenases generally we decided to examine the effects of alloputiol and oxypurinol on the highly purified xanthine dehydrogenase (EC 1.2.1.37) from turkey liver.

On the reaction of formaldehyde with turkey liver xanthine dehydrogenase

Abstract 1. 1. Turkey liver xanthine dehydrogenase, whether native or modified by pre-treatment with cyanide, arsenite or methanol, is progressively inactivated by formaldehyde. 2. 2. Such inactivation is complete with respect to xanthine oxidation but incomplete in the case of NADH diaphorase activity. 3. 3. Formaldehyde-treated enzyme has an altered visible absorption spectrum, and is less readily reduced by NADH or re-oxidized by oxygen following reduction than is native enzyme. 4. 4. It is concluded that the effects of formaldehyde result from its binding to the molybdenum centres and to one or more additional sites on the enzyme.

Further observations on the effects of S-triazine derivatives on purine metabolizing enzymes.

Abstract 1. 1. Various s-triazine derivatives completely inhibit the turkey liver xanthine dehydrogenase catalysed oxidation of xanthine. K i values show that the relative effectiveness of these compounds is as follows: ammeline > ammelide > allantoxaidin > cyanuric acid > melamine. Oxonate, a related triazine derivative, is without effect on this enzyme. 2. 2. The differential effects of ammelide on the NADH diaphorase activities of the turkey enzyme indicate that the oxidizing substrates dichlorophenol indophenol and trinitrobenzene sulphonate inter- act with the molybdenum prosthetic groups. 3. 3. Attempts at purifying urate oxidase by affinity chromatography using oxonate (a potent inhibitor of this enzyme) coupled to aminopropanyl-Sepharose were unsuccessful.

Some properties of a multiple-form β-galactosidase from an Arthrobacter SP.

Abstract 1. 1. β-Galactosidase from Arthrobacter sp. has been purified to apparent homogeneity. It has a mol. wt in the region of 500,000, a pH optimum of 6.3–6.4 and Michaelis constants of 0.21 mM and 10.5 mM for o-nitrophenyl-β-D-galactopyranoside and lactose, respectively. 2. 2. The metal dependency of the enzyme resembles that of Escherichia coli β-galactosidase, but it is less stable to heat and urea than the E. coli enzyme. 3. 3. Zn2+ inhibition of the enzyme is suppressed in the presence of 20 mM phosphate. 4. 4. Crude extracts of Arthrobacter sp. contain several electrophoretically distinct forms of β-galactosidase.