THE STEPWISE HYDROLYSIS OF ADENINE-NUCLEOTIDES BY ECTOENZYMES OF RAT RENAL BRUSH-BORDER MEMBRANES

被引:40
作者
CULIC, O
SABOLIC, I
ZANICGRUBISIC, T
机构
[1] UNIV ZAGREB, FAC PHARM & BIOCHEM,DEPT MED BIOCHEM,A KOVACICA 1, POB 156, YU-41000 Zagreb, YUGOSLAVIA
[2] UNIV ZAGREB, FAC MED, DEPT PHYSIOL, YU-41000 Zagreb, YUGOSLAVIA
关键词
(Rat kidney); Adenosine; Brush-border membrane vesicle; Ectoenzyme; Ectonucleotidase;
D O I
10.1016/0005-2736(90)90249-N
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg 2+ < Ca 2+ < Mn 2+ < Ba 2+ < Zn 2+ . At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg 2+ -stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity (K′ m = 0.16 mM) and low-affinity site (K′ m = 9.0 mM, respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent K m = 0.38 ± 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent K m = 76 ± 9 μM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems. © 1990.
引用
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页码:143 / 151
页数:9
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