Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

Abstract

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200-400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40-60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 /UM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5-10 JUM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.

Intracellular calcium storage and release in the human platelet. Chlorotetracycline as a continuous monitor.

“…1 fiM. This agrees with an apparent K m of 0.5-1 /iM determined for isolated dense tubules (Robblee et al, 1973;Kaser-Glanzman et al, 1977). The line through the points was fitted according to a second-power dependence of the rate on…”

“…The sources of intracellular sequestered Ca ++ , and the regulation of its release and uptake are not well understood. Biochemical studies have identified and characterized an ATP-dependent Ca ++ uptake mechanism in human platelet membrane fractions (Robblee et al, 1973;Kaser-Glanzman et al, 1977;Fox et al, 1979;Javors et al, 1982). A fraction of the membranes was purified and identified as the dense tubular system (Meriashi et al, 1981) and active uptake by this fraction was shown to be inhibited by trifluoperazine (TFP).…”

Control, modulation, and regulation of cell calcium

“…Little or no effect of phosphate Moore et al 1974Moore et al , 1975Selingeret al t970 Oh tsuki 1969;DeMeis et al 1970;Selinger et al 1970;Alonso et al 1971 ;Moore et al 1974;Baumrucker and Keenan 1975;Moore andPastan 1977a, 1978;Farber et at. 1977;Watson and Siegel 1978Moore et al 1974Moore and Pastan 1978;AIonso et al 1971Otsuka et al 1965Robinson and Lust 1968;Ohtsuki 1969;DeMeis et al 1970;AIonso et al 1971 ;Robblee et al1973;Moore et al 1974Moore et al , 1975Moore and Pastan 1977a;Bruns et al 1976;Farber et al 1977DeMeis et al 1970Alonso et al 1971 ;Robblee et al 1973;Moore et al 1974Moore et al , 1975Moore and Pastan 1977a;Trotta and DeMeis 1975 ;Farber et al 1977Moore et al 1975Bruns et at. 1976;Moore and Pastan 1977a;Farber et al 1977;Bygrave 1978Alonso et al 1971Moore et al 1974Moore et al , 1975…”

“…1977;Watson and Siegel 1978Moore et al 1974Moore and Pastan 1978;AIonso et al 1971Otsuka et al 1965Robinson and Lust 1968;Ohtsuki 1969;DeMeis et al 1970;AIonso et al 1971 ;Robblee et al1973;Moore et al 1974Moore et al , 1975Moore and Pastan 1977a;Bruns et al 1976;Farber et al 1977DeMeis et al 1970Alonso et al 1971 ;Robblee et al 1973;Moore et al 1974Moore et al , 1975Moore and Pastan 1977a;Trotta and DeMeis 1975 ;Farber et al 1977Moore et al 1975Bruns et at. 1976;Moore and Pastan 1977a;Farber et al 1977;Bygrave 1978Alonso et al 1971Moore et al 1974Moore et al , 1975Trotta and DeMeis 1971 ;Brans et al 1976;Moore and Pastan 1977a;Farber et al 1977Selinger et al 1970Moore et al 1974Moore et al , 1975Moore andPastan 1977a, b, 1978;Blaustein et al 1978c Robinson andLust 1968;…”

Calcium-activated ATPase activity in a plasma membrane-rich preparation of rat pancreas

“…In several membrane preparations, active calcium extrusion has been linked to plasma membrane-bound calcium-activated ATPases (Schatzmann & Vincenzi, 1969 ;Martonosi, 1969;Makinose, 1972;Robblee, Shepro & Belamarich, 1973). This, however, because of the non-uniform secretagogue effect on the enzyme activity, would not appear to be the case in the pancreas.…”