Studies on a Ca2+-dependent nucleoside triphosphate pyrophosphohydrolase in rat liver plasma membranes.
A membrane-bound Ca2+-dependent nucleoside triphosphate pyrophosphohydrolase was solubilized in deoxycholate, separated from inorganic pyrophosphatase, and partially characterized. The Km for a variety of substrates was determined. At 10(-4) M free Ca2+ (pH 8.0) the Km values for ATP and GTP were 0.32 and 2.2 microM, respectively. With ATP as substrate, Mg2+, Sr2+, and Ba2+ could only replace Ca2+ to a limited degree. Both purine and pyrimidine nucleoside triphosphates were hydrolyzed yielding PPi and mononucleotides and similarly AMP and formed from adenosine-(beta gamma-methylene)triphosphate. UDPglucose was hydrolyzed at the pyrophosphate bond. Tripolyphosphate and phosphoribosyl-1-pyrophosphate (P-rib-PP) were not hydrolyzed. Substrate competition experiments showed that GTP inhibited pyrophosphohydrolysis of ATP competitively. However, UDP glucase and adenosine-(beta gamma-methylene)triphosphate inhibited ATP pyrophosphohydrolysis in a non-linear manner. Adenosine-(beta gamma-methylene)triphosphate inhibited pyrophosphohydrolysis of UDPglucose non-competitively, whereas UDPglucose inhibition of adenosine-(beta gamma-methylene) triphosphate pyrophosphohydr-lysis was competitive. The molecular weight of ATP pyrophosphohydrolase was estimated at 120 000 and the pI at 5.1 Pyrophosphohydrolysis of adenosine-(beta gamma-methylene)triphosphate was studied in a number of rat organs. Nearly all activity could be sedimented at 50 000 X g. Very high activities were found in liver, kidney and small intestine, whereas low activities were found in brain and blood.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011755:Pyrophosphatases / Q000378:metabolism*; D051381:Rats; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D002413:Cations, Divalent; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C021895:nucleoside triphosphate pyrophosphatase; D014532:Uridine Diphosphate Glucose