5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity.
An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.
Lee
R S
RS
Department of Pathology, Wellington School of Medicine, New Zealand.
Ford
H C
HC
D015720:5'-Nucleotidase; D000227:Adenine Nucleotides / Q000494:pharmacology; D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology*; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014327:Trophoblasts / Q000201:enzymology*
D000227:Adenine Nucleotides; D002413:Cations, Divalent; D003035:Cobalt; D009708:Nucleotidases; D015720:5'-Nucleotidase; D011755:Pyrophosphatases; C023417:FAD pyrophosphatase