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Enzymic characteristics of the isoenzymes of rat epididymal neutral alpha-mannosidases and their changes during development in vivo.

ABSTRACT: Three isoenzymic species of neutral alpha-mannosidase (I, II and III) were partially purified from rat epididymis and characterized. Calcium phosphate gel preferentially adsorbed acidic alpha-mannosidase activity, thereby removing the acidic enzyme from the neutral mannosidases. The neutral enzymes, which are of cytosolic origin, require Co2+ for activity, and Mn2+ can substitute partially for Co2+. Mg2+ or Ca2+ had little effect on the activity of the isoenzymes, whereas Zn2+ (100 microM) was a potent inhibitor of the mannosidases. The Km values of mannosidases I, II and III for Co2+ were 10, 10 and 2.7 mM respectively. There was marked alteration of the specific activity of the neutral alpha-mannosidases during epididymal development in vivo. The specific activities of mannosidases I and II were relatively high in the young (24 days) rats, and during the subsequent development the specific activities decreased markedly (approx. 2-3-fold). On the contrary, mannosidase III, which showed relatively low specific activity during 24-45 days of age, increased markedly (approx. 2-fold) as the animals reached adulthood.

SUBMITTER: Dutta P

PROVIDER: S-EPMC1153364 | biostudies-other | 1984 Mar

REPOSITORIES: biostudies-other

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Project description:Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an omega-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca(2+) content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca(2+) and with an acidic pH, Ca(2+) had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate.

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Enzymic characteristics of the isoenzymes of rat epididymal neutral alpha-mannosidases and their changes during development in vivo.

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