Biochemical evidence for Mn2+-dependent 5'-nucleotidase activity in isolated rod outer segments.
Recently cytochemical evidence has been presented for a novel enzyme activity, i.e. 'manganese-dependent pyrimidine 5'-nucleotidase (MDPNase)' activity in the rod outer segments (ROS) of rat retinas in situ and in isolated rat ROS. The present biochemical study was undertaken to seek further evidence for this enzyme activity using an independent method. A series of enzyme assays was carried out to test for MDPNase activity in Triton extracts of rat ROS isolated by sucrose density gradient centrifugation. Hydrolysis of the substrate, cytidine-5'-monophosphate, was measured spectrophotometrically and expressed as microgram of released inorganic phosphorus hr-1 mg-1 protein in the sample. The results showed that the ROS extracts contained enzyme activity (18.1 +/- 3.8) that was increased 5-6-fold (102.3 +/- 10.6) in the presence of 7.4 mM MnCl2. The enzyme activity was not enhanced by Mg2+ ions (19.0 +/- 7.7) and was strongly inhibited by 10-20 mM NaF (11.8 +/- 2.9). Assays for substrate specificity revealed that the Mn2+-stimulated phosphatase activity was specific for 5'-nucleotides. Pyrimidine nucleotides (5'-CMP and 5'-UMP) were the preferred substrates. Comparison of enzymatic hydrolysis of 5'-CMP and 5'-AMP over a pH range from 4.5 to 8.0 revealed that at acid pH, the majority of the observed 5'-nucleotidase activity (82% at pH 5.0, 58% at pH 5.5) was manganese dependent, whereas at neutral pH and above, most of the enzyme activity was unaffected by the presence of Mn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Irons
M J
MJ
Department of Anatomy, University of Toronto, Ontario, Canada.
O'Brien
P J
PJ
D015720:5'-Nucleotidase; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002712:Chlorides; D003568:Cytidine Monophosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010786:Photoreceptor Cells / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D002712:Chlorides; D017895:Manganese Compounds; D000249:Adenosine Monophosphate; D008345:Manganese; D009708:Nucleotidases; C025221:cytidine-5'-monophosphatase; D010744:Phosphoric Monoester Hydrolases; D015720:5'-Nucleotidase; D003568:Cytidine Monophosphate; C025340:manganese chloride