Michiya Fujita

Differential isolation of microvillous and basolateral plasma membranes from intestinal mucosa: Mutually exclusive distribution of digestive enzymes and ouabain-sensitive ATPase

Abstract Using density-gradient centrifugation microvillous membranes were isolated from a partially purified brush border fraction, and basolateral plasma membranes from a crude mitochondrial fraction. Alkaline p- nitrophenyl phosphatase, sucrase and leucylglycine hydrolase were enriched in the former, and ouabain-sensitive ATPase in the latter. Electron microscopy showed that the microvillous membrane fraction was composed of thick membrane vesicles of regular size, and the basolateral membrane fraction of vesicles more irregular in shape and size. Most of the work was performed with mouse jejunum. Similar results were obtained with rat.

Potassium-ion stimulated p-nitrophenylphosphatase activity occurring in a highly specific adenosine triphosphatase preparation from rabbit brain

Abstract 1. 1. p -Nitrophenylphosphatase activity was detected in a highly specific Na + - and K + -dependent ATPase preparation obtained by NaI treatment of rabbit-brain microsomes. 2. 2. The p -nitrophenylphosphatase activity required K + and was inhibited by Na + . The concentrations of K + and Na + required for half maximal activation and half maximal inhibition were 3.2 and 32 mM, respectively. 3. 3. The p -nitrophenylphosphatase activity was inhibited by ouabain, and this inhibition was not affected by Na + . The concentration of ouabain required for 50% inhibition of p -nitrophenylphosphatase activity was approx. 2.5·10 −5 M, which was 30 times that required for 50% inhibition of ATPase activity. However, the former was decreased to 2.5·10 −6 M by the addition of 40 μM of ATP. 4. 4. In the absence of Na + the preparation was active exclusively with -nitrophenylphosphate. None of the following was hydrolyzed: ATP, AMP, 2′-(3′)-AMP, CMP, IMP, GMP, glucose 6-phosphate, glucose 1-phosphate, β-glycerophosphate or phenylphosphate. 5. 5. The p -nitrophenylphosphatase activity was inhibited by a low concentration of ATP. Inhibition was of the simple non-competitive type with a K i value of 4·10 −5 M. The inhibition of ATPase activity by p -nitrophenylphosphate was also of the non-competitive type, the K i for p -nitrophenylphosphate being 4·10 −2 M. 6. 6. The K m for p -nitrophenylphosphate was 1.5·10 −3 M, and that for ATP, 6·10 −5 M. 7. 7. p -Nitrophenylphosphatase activity was inhibited by 60–80% with 3 mM of any of the following: GTP, UTP, CTP, ADP, AMP, PP i , or P i . 8. 8. ATPase and p -nitrophenylphosphatase were similarly inactivated by either thermal denaturation or trypsin treatment. 9. 9. There was a fairly good correlation between the inhibition curves for p -nitrophenylphosphatase and ATPase in the presence of increasing concentrations of N -ethylmaleimide or diisopropylphosphorofluoridate (DFP). The inhibition of both activities by DFP was markedly enhanced by K + , and the activities were protected against the inhibition by either p -nitrophenyphosphate or ATP. 10. 10. It is suggested that the two activities could be attributed to at least two different active sites which, however, appear to be closely correlated.

Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal mucosa

Abstract A careful washed preparation of intestinal brush border membranes from rat contained two-thirds of the sucrase activity of a starting homogenate and 2–4% of the ouabain-sensitive ATPase activity.

Protein components of two different regions of an intestinal epithelial cell membrane: Regional singularities

Abstract The two different regions of the plasma membrane, i.e. apical and basolateral membranes, of intestinal epithelial cells were analyzed as to their proten components. They showed very contrasting profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apical membranes possessed several major components with apparent molecular weights larger than 108 000, most of which were also periodic acid-Schiff reagent positive. In contrast, there were no protein components with corresponding molecular weights in the basolateral membrane. The electrophoretic profile of the latter was strinkingly simple. The dominant band was assigned a molecular weight of 101 000 and was periodic acid-Schiff reagent negative. No major components were shared by the two membranes.

On the possible role of the phosphorylated intermediate in the reaction mechanism of (Na+−K+)-ATPase

Abstract 1. Phosphorylation and dephosphorylation of (Na + −K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) by [γ- 32 P]ATP was examined using highly specific NaI-treated preparations from pig-brain microsomes. 2. The phosphorylated enzyme consisted of an essentially single chemical species. The incorporated phosphate was relatively stable in an acidic medium (pH 1–2), and was easily released from the enzyme by treatment with hydroxylamine at a neutral pH. 3. The chemical nature of the phosphate bond was similar under the following alternative conditions: (a) whether the labeling was stopped by HClO 4 or dodecyl sulfate; (b) whether K + was present or not in addition to Na + ; or (c) whether the labeling was performed at 37° or 0°. 4. Longer incubation of the enzyme preparation with [γ- 32 P]ATP resulted in another form of incorporation which was hydroxylamine-resistant. 5. The high specificity of the ATP-hydrolyzing reaction of (Na + −K + )-ATPase to Na + and K + was partially lost at 0°, while the specific response of the phosphorylated enzyme to Na + and K + was still maintained. 6. Based on these results, possible relationships of the phosphorylated (Na + −K + )-ATPase to the ATP-hydrolyzing reaction of the enzyme are discussed.

Molecular weight of Na, K-ATPase approximated by the radiation inactivation method.

Abstract The Na, K-ATPase has not yet been purified as a single molecular species, mainly because of its insolubility. Nevertheless, an approximate molecular weight of the dried enzyme can be obtained by the radiation inactivation technique introduced by Hutchinson and Pollard (1961) . Using this procedure Kepner and Macey (1966) reported a value of about one million as the molecular weight for the ATPase of freeze-dried human erythrocyte ghosts. Their preparation contained both the ouabain sensitive and insensitive ATPase. A reinvestigation of this problem seemed worthwhile in that our preparation of Na, K-ATPase from brain microsomes is highly specific, essentially free of the ouabain-insensitive enzyme ( Nakao et al. , 1965 ). This preparation showed that the phosphorylated protein, obtained in a reaction with ATP 32 requiring both Na and Mg ions, consisted of a single chemical species as indicated by the time course of hydrolysis and by its sensitivity towards hydroxylamine. In this report we have estimated the target molecular weight of the enzyme involved in the phosphorylation reaction with ATP and in the overall hydrolysis of ATP.

Approximation of molecular weight of (Na+-K+)-ATPase

Abstract 1. 1. A (Na+-K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparation from pig brain microsomes was disrupted into smaller particles by sonication in the presence of a detergent. A portion of the dissected protein was active, completely inhibited by ouabain, and remained in the supernatant after centrifugation at 100 000 × g. A portion of the supernatant activity was retarded over a wide range in gel filtration through a Sepharose 4B column. Fractions of retarded activity were eluted at the corresponding positions in rechromatography through the same column. 2. 2. The partial specific volume of the eluted active protein was determined as 0.85 by means of a sort of equilibrium centrifugation in sucrose media with several different densities. 3. 3. Sucrose gradient centrifugation was performed using 2 fractions of the eluted active protein and their molecular sizes were estimated. 4. 4. Agreement was obtained regarding the smallest molecular size of active (Na+-K+)-ATPase protein estimated by various methods. The values also roughly coincided with the estimated molecular size in the target bombardment experiments reported previously by ourselves. 5. 5. The possibility of application of the techniques adopted here to other unpurified protein preparations was discussed.

Membrane fractions of rabbit granulocytes. 1. The presence of ouabain-sensitive ATPase and the electrophoretic patterns

In one of two membrane fractions isolated from the homogenate of casein-induced rabbit granulocytes ouabain-sensitive ATPase activity was found definitely. Electrophoretic patterns of these membrane fractions showed no significant differences in staining for carbohydrate and protein.