Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Abstract

Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant, Km (1.5 x 10(5) M), similar to the inhibitory constant KI (3 X 10(-5) M), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabaiation rate constants reported for other tissues and species. The high dissociation rate constant 3.6 x 10(-2) sec-1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represents a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.

Na, K-ATPase and the development of Na+ transport in rat distal colon

“…This suggests that factors within the cell and/or cell membrane may control the turnover. This is consistent with previous findings of higher catalytic turnover number in more purified membrane preparation of intestinal Na,K-ATPase [19]. Using the data from this study, the maximum turnover rate for Na § in intact epithelium can be estimated by the following equation: turnover rate = 3[(I~/F) 9 N 9 60]/Na,K-pump density (5) where F and N are Faraday's constant and Avogadro's number.…”

“…The value of KI is identical to K~ previously reported in the distal colon of adult rats [15]. The values ofK D are similar to the KD identified in the membrane-rich fraction of colonocytes and jejunal enterocytes from adult rats [19,20]. In the colon and small intestine of the rabbit, which is more sensitive to ouabain than the rat, KD was found to be lower [31,32].…”

“…The age-dependent increase in the number of Na,K pumps suggests that a similar increase in Na,K-ATPase activity is due to a change in the number of pump sites. The catalytic turnover number is within the range reported for other tissues [19] and very similar to the values of rabbit colon [31] and rabbit collecting tubule [10], which is an epithelium with transport properties similar to those of colon. However, all of these values are much smaller than the value 10,000 per min usually quoted for purified Na,K-ATPase.…”

Epithelial potassium transport: Tracer and electrophysiological studies in choroid plexus

“…The values obtained for Kin, kl, k_ 1 are intermediate between those reported for human erythrocytes (Erdmann and Hasse, 1975) and rabbit renal tubule (Shaver and Stifling, 1978) at 22-25 ~ (see also Harms & Wright, 1980), Using the values estimated for the kinetics of ouabain binding to the choroid plexus, it is possible to calculate the half time for ouabain binding at any This is considerably faster than that shown in Fig. 6, but it is consistent with the observation that 1 x 10-4 ouabain eliminates the electrogenic component of the membrane potential in 7 sec (Fig.…”

Ion permeability of rabbit intestinal brush border membrane vesicles

“…The first was from cell counts of photomicrographs of small intestine. There were approximately 108 cells/cm 2 (Harms & Wright, 1980). The second method involved measurement of the amount of mucosal protein scraped off a known serosal surface area of intestine (6mg/cm 2) and the amount of protein per cell (~4 x 10 .7 mg/cell; Harms, personal communication).…”