Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

Knauf, Philip A.; Proverbio, Fulgencio; Hoffman, Joseph F.

doi:10.1085/jgp.63.3.305

Cited by 75 publications

(35 citation statements)

References 33 publications

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“…First, we have demonstrated that the phosphorylated intermediate of the Na/K-ATPase of intestinal basal lateral membranes has the same apparent mol wt as that identified in human red cells (Knauf, Proverbio & Hoffman, 1974) and as that isolated from dog kidney (Kyte, 1971), rabbit, pig, or sheep kidney (Jorgensen, 1975), brain (Uesugi et al, 1971), and shark rectal gland (Hokin et al, 1973). Secondly, we have shown that the phosphoprotein of the Na/K-ATPase of the basal lateral membrane is an acyl phosphate by its sensitivity to hydroxylamine.…”

Section: Discussionmentioning

confidence: 81%

“…Membranes were phosphorylated by the method of Knauf et al (1974). Membranes were phosphorylated on ice for 15 sec in a medium containing 5 mM Tris-Hepes pH 7.5, MgC12 12 gM, ATP 7 a2p 0.4-10 gM, and NaC1 50 raM.…”

Section: Phosphorylationmentioning

confidence: 99%

“…From a densitometric tracing of Coomassie Blue stained gels of basal lateral membranes purified 10 fold, 15% of the basal lateral membrane protein has an apparent mol wt of 100,000. To determine whether the Coomassie staining protein and the phosphorylated intermediate were identical, we followed the strategy of Knauf Proverbio, and Hoffman (1974) of digestion of the intact cells by pronase. Isolated cells were incubated at 37 ~ for 10 rain with and without pronase 25 pg/ml.…”

Section: Pronase Digestionmentioning

confidence: 99%

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

Harms

Wright

1980

J. Membrain Biol.

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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.

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Section: Discussionmentioning

confidence: 81%

Section: Phosphorylationmentioning

confidence: 99%

Section: Pronase Digestionmentioning

confidence: 99%

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

Harms

Wright

1980

J. Membrain Biol.

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“…Incorporation of 32p from y-[32p]-ATP into isolated cortices was performed essentially as described by Knauf, Proverbio and Hoffman (1974). Cortices were obtained by differential centrifugation of homogenized oocytes in Ca++-free De Boer's solution (Richter & Tintschl, 1983).…”

Section: Methodsmentioning

confidence: 99%

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation ofXenopus oocytes

1984

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During progesterone-stimulated maturation of defolliculated full-grown Xenopus oocytes, the activities of the transport systems for L-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms. A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8-12 X 10(9) molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K+ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na-K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na-K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg++ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na+-dependent incorporation of 32P from gamma-[32P]-ATP that can be reversed by the addition of K+. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.

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“…These interactions are expressed by both stimulation and inhibition of activities, the end result being a complex function of the cations involved, their concentrations, as well as the nature and concentrations of other ligands simultaneously present. For instance, Ca ions, in the presence of Mg are inhibitors of Na,K-ATPase (Skou, 1957; Dunham & Glynn, 1961;Epstein & Whittam, 1966; Tobin, Akera, Baskin & Brody, 1973; Knauf, Proverbio & Hoffman, 1974;Fukushima & Post, 1978;Huang & Askari, 1982; Beauge' & Campos, 1983) and the Na pump (Brown & Lew, 1983). This effect is more marked on (Na + K)-dependent than on Na-dependent activity and at high than at low ATP concentrations (Beauge & Campos, 1983), and is exerted by affecting the external-K-stimulated dephosphorylation as well as enzyme phosphorylation (Tobin et al 1973;Fukushima & Post, 1978;Beauge & Campos, 1983).…”

Section: Introductionmentioning

confidence: 99%

Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K‐ATPase.

Beaugé¹,

Campos²

1986

The Journal of Physiology

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SUMMARY1. Interactions ofNa, K, Ca and Mg ions with partially purified Na,K-ATPase from pig kidneys were investigated using as a tool simultaneous determinations of adenosine 5'-triphosphate (ATP)-adenosine 5'-diphosphate (ADP) exchange and ATPase activity catalysed by the enzyme.2. In the presence of 120 mM-NaCl and 0 5 mM-MgCl2, the effects 12 mM-KCl on ATP-ADP exchange depended on the ATP and ADP concentrations: stimulation (about 10-fold) with 3 mM-ATP-075 mM-ADP, no effect with 0-04 mM-ATP-0 01 mM-ADP and inhibition when ATP and ADP concentrations were 0 003 mm and 0-001 mm respectively. The apparent affinities (KO.5) for K stimulation of ATP-ADP exchange and Na,K-ATPase activity were indistinguishable from each other both at 20 mmand 120 mM-Na. All Na and Na + K-dependent exchanges were completely abolished by 10-4 M-ouabain.3. In the absence of K, intermediate Na concentrations were inhibitory of ouabain-sensitive ATP-ADP exchange and ATPase activity. This Na inhibition was more pronounced at low than at high ionic strength and was more noticeable in the exchange reaction. K ions antagonized these Na effects; this K-Na antagonism was more effective on ATPase than on the ATP-ADP exchange.4. Ca ions, in the absence of Mg, could sustain ATP-ADP exchange. Qualitatively the behaviour of the exchange reaction was similar to that observed with Mg. Quantitatively the rates were always lower with Ca. In the presence ofMg, at low ionic strength and at concentrations which stimulated phosphatase activity, Ca ions were not able to counteract Na inhibition of ATPase and ATP-ADP exchange or to stimulate ATPase activity in the presence of Na. This is another indication of the different reactivity of the K sites involved in phosphatase and ATPase activities as well as of those responsible for the release of Na inhibition of ATPase and exchange reaction.5. In K-free solutions containing 0-5 mM-Na and low ionic strength, Mg was a powerful inhibitor of ouabain-sensitive ATPase and ATP-ADP exchange; the curves relating remaining activities to Mg concentrations were identical for both reactions. When Na was increased to 5 mM, ATP-ADP exchange became more sensitive and ATPase more resistant to Mg inhibition. These data are consistent with (i) a Na-Mg antagonism at the phosphorylation step, and (ii) a Na-Mg synergism in blocking the 1 PHY 375ATP-ADP EXCHANGE AND Na,K-ATPase E2PNa-E1PNa transformation. At high ionic strength, with higher concentrations of Na alone and of Na + K, Mg was also a strong inhibitor of ATP-ADP exchange; in this case the possible mechanism is less clear, although K ions could, to some extent, counteract this Mg effect. 6. The present results are consistent with Na inhibition of ATP-ADP exchange and ATPase activity by stabilizing the E2P conformation. This stabilization is selective in that it is easier to overcome in the forward direction (towards ATP hydrolysis) than in the backward directions (leading to ATP synthesis). These results seem to be best explained on the basis that K stimulation of ouabain...

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Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

Cited by 75 publications

References 33 publications

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

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

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation ofXenopus oocytes

Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K‐ATPase.

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