Calcium transport in inside-out membrane vesicles prepared from rabbit neutrophils.

M Volpi,P H Naccache,R I Sha'Afi

doi:10.1016/s0021-9258(18)32598-5

Abstract

Inside-out membrane vesicles capable in the presence of MgATP of accumulating calcium and containing Ca2+-ATPase activity have been prepared from rabbit neutrophils. The transport system has a Km for Ca2+ of 2.8 +/- 0.3 microM and a Vmax of 5.3 +/- 0.3 nmol/mg of protein . min and a Km for ATP of 0.35 +/- 0.13 mM and a Vmax of 5.8 +/- 0.3 nmol/mg protein . min. The Ca2+-activated ATPase system has a Km for Ca2+ of 0.2 +/- 0.05 microM and a Vmax of 4.0 +/- 0.5 nmol/mg protein . min, and a Km for ATP of 0.36 +/- 0.2 mM and a Vmax of 5.0 +/- 0.6 nmol/mg protein . min. It was also found that neither these inside-out vesicles nor the intact rabbit neutrophils contain a Na+/Ca2+ exchange mechanism.

Highlights

Inside-out membranveesiclescapable in the presenceVery little is known about the kinetic characteristics of this of MgATP of accumulatingcalcium and containing pump, its role in the regulation of intracellular calcium, and
Activation of the neutrophils begins with the binding of the stimulus to specificplasma membrane-located receptor sites
We have recently reported on the preparation of inside-out membrane vesicles from neutrophils capable of actively transporting calcium [7]

Summary

MATERIALS AND METHODS

Is a transientincrease in the intracellular level of one or more Inside-out plasma membrane vesicles from rabbit neutrophils were of the so-called “second messengers,” which is thought prepared by inducing the cells to ingest parafin oil droplets. Thereafter, the cells were washed twice by resuspending them in 40 ml of cold saline solution (150mM NaCl, pH 7.0), followed by centrifugation at 1,200X g for 2 and to the plasma membrane [1].The presence of a specific min. The cells were resuspended in the modified Hanks‘ buffer calcium pump driven by the hydrolysis of ATP by a containing 1 mg/ml dextrose at a concentration of lo cell/& and (Mg2’,Ca2+)-activatedATPase located in the plasma mem- were incubated in a rotary shaker water bath for 15 min at 37 “C.

Calcium Transport in Neutrophils

RESULTS

IS m I oI s

Calcinium Transport Neutrophils

Findings

DISCUSSION