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Two-step internalization of Ca/sup 2 +/ from a single E approx. P x Ca/sub 2/ species by the Ca/sup 2 +/-ATPase

Journal Article · · Biochemistry; (United States)
OSTI ID:7000152
;

Phosphorylation by ATP of E x *Ca/sub 2/ (sarcoplasmic reticulum vesicles (SRV) with bound /sup 45/Ca/sup 2 +/) during 5-10 ms leads to the occlusion of 2 *Ca/sup 2 +/EP/sub tot/ (quench by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone) in both empty (10 ..mu..M free Ca/sup 2 +//sub in/) or loaded SRV (20-40 mM free Ca/sup 2 +//sub in/). The rate of Ca/sup 2 +/ internalization from the occluded E approx. P x *Ca/sub 2/ was measured by using ADP + EGTA quench; a Ca/sup 2 +/ ion that is not removed by this quench is defined as internalized. These data show that the two Ca/sup 2 +/ ions are internalized sequentially, presumably from separate sequential sites in the channel. (/sup 32/P)EP x Ca/sub 2/ obtained by rapid mixing of E x Ca/sub 2/ with (..gamma..-/sup 32/P)ATP and EGTA disappears in a biphasic time course with a lag corresponding to approx. 34 s/sup -1/, followed by EP* decay with a rate constant of approx. 17 s/sup -1/. This shows that both Ca/sup 2 +/ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of /sup 45/Ca/sup 2 +/ internalization from 34 to 69 s/sup -1/ for the first Ca/sup 2 +/ and from 17 to 34 s/sup -1/ for the second Ca/sup 2 +/. High (ATP) also accelerates both phases of (/sup 32/P)EP x Ca/sub 2/ disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approx.P x Ca/sub 2/ that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 ..mu..Lmg, so that one turnover of the enzyme gives 4 mM internal (Ca/sup 2 +/).

Research Organization:
Brandeis Univ., Waltham, MA (USA)
OSTI ID:
7000152
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:8; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
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ALKALINE EARTH ISOTOPES
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BIOCHEMICAL REACTION KINETICS
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CALCIUM ISOTOPES
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CHARGED PARTICLES
DAYS LIVING RADIOISOTOPES
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ODD-ODD NUCLEI
ORGANIC COMPOUNDS
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PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM