Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid.
A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.
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Department of Biochemistry, National University of Singapore, Kent Ridge Crescent.
Teo
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Thiyagarajah
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D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000493:pharmacokinetics*; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010306:Parotid Gland / Q000201:enzymology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D014268:Trifluoperazine; D000255:Adenosine Triphosphate; D012964:Sodium; D011188:Potassium; D002118:Calcium