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 · Tue Apr 19 04:00:00 EDT 1988 · Biochemistry; (United States)

OSTI ID:7000152

Khananshvili, D; Jencks, W P

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

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METAL COMPOUNDS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CALCIUM 45
CALCIUM COMPOUNDS
CALCIUM ISOTOPES
CATIONS
CELL CONSTITUENTS
CHARGED PARTICLES
DAYS LIVING RADIOISOTOPES
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
HYDROLASES
INTERMEDIATE MASS NUCLEI
IONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MEMBRANE TRANSPORT
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM