Sjoerd L. Bonting

Studies on sodium-potassium-activated adenosine triphosphatase: I. Quantitative distribution in several tissues of the cat

Abstract The quantitative distribution of sodium-potassium-activated adenosine triphosphatase, which had been shown to play a role in active transport of sodium and potassium in erythrocytes, has been determined in 36 tissues of the cat and in human retina, ciliary body, and erythrocytes. The enzyme was found in 29 of the 36 cat tissues and in the three human tissues. The activity ranged from 1.52 in cat gray matter to 0.00027 in cat erythrocytes, expressed in millimoles of adenosine triphosphate split/g. wet weight of tissue/hr. at 37 °C. Highest activities were noted in nervous tissue and in tissues concerned with secretory function. No in vitro effects on the enzyme in cat gray matter and retina were observed with 13 hormones and five other physiologically important compounds.

Studies on sodium-potassium-activated adenosinetriphosphatase. V. Correlation of enzyme activity with cation flux in six tissues.

Abstract The activity of the enzyme Na-K-activated ATPase, which is closely related to the active transport system of sodium and potassium in human erythrocytes, was determined in human erythrocytes, frog toe muscle, squid giant axone, frog skin, toad bladder, and in the noninnervated membrane of the Sachs organ of the electric eel. The temperature coefficient of the enzyme was determined in each tissue except the erythrocytes. The results were expressed in moles/sq. cm./sec. and compared with the active cation fluxes, expressed in the same units, for these tissues reported by other investigators. The enzyme activities were corrected for the temperature, at which the flux values had been obtained. A significant correlation was found between Na-K-activated ATPase activity and active cation flux over a 25,000-fold range. The average ratio of equivalents cation transported per mole ATP hydrolyzed (cation/ATP ratio) was 2.56 ± 0.19, which is significantly higher than 2, but not significantly different from 3. The total ATPase activity was not significantly correlated with the flux values. The value of 2.56 for the cation/ATP ratio agrees with many other determinations of this ratio for various tissues reported in the literature. These findings further strengthen the assumption that the Na-K-activated ATPase is closely related to the active cation transport involved in maintenance of ion gradients in single cells, transport of salt and water across epithelial membranes, and repolarization processes in muscle, nerve, and electric organs.

Studies on sodium-potassium-activated adenosinetriphosphatase. IV. Correlation with cation transport sensitive to cardiac glycosides

A systematic study was made of the occurrence in tissues with a cardiac glycoside-sensitive cation transport system of the enzyme sodium-potassium-activated adenosinetriphosphatase (Naue5f8K ATPase), which has been shown to function in the active transport of sodium and potassium in erythrocytes and to be specifically inhibited by cardiac glycosides. n nThe enzyme was found to be present in significant quantities in all 21 tissues from 10 species studied. The activities ranged from 1.24 in human erythrocytes to 1825 in guinea pig brain, expressed in millimoles of adenosine triphosphate split/kg. wet weight of tissue/hr. at 37 °C. n nIn 8 out of 12 tissues, where the cardiac glycoside-sensitive cation exchange rate could be calculated, a qualitative correlation was found between the cation exchange rate and the Naue5f8K ATPase activity. n nInhibition curves were determined for the Naue5f8K ATPases of four different tissues from four species as a function of ouabain concentration. The slopes of the curves differed considerably, and the half-maximal inhibition concentrations varied over a 17-fold range. There was both a species effect and a tissue effect. The results were discussed from the point of view of the in vivo use of cardiac glycosides for the demonstration of Naue5f8K ATPase involvement in a physiological process. n nThe distribution of Naue5f8K ATPase activity in centrifugal fractions of rat liver and brain homogenates was determined. In liver almost all activity was present in the “nuclear” fraction, while in brain the activity was spread over all three particulate fractions. No activity was found in the supernatant fractions. This pattern is consistent with predominant localization of the enzyme in the cell membranes of both tissues.

Determination of microgram quantities of deoxyribonucleic acid and protein in tissues grown in vitro.

Summary 1. A simple and accurate procedure is described for the routine determination of deoxyribonucleic acid and protein in tissue culture material, microtome sections, or other histologically defined tissue samples. 2. The tissue is dissolved by incubation in 1 N NaOH for 1 hr. at 37°C. Aliquots of the alkaline digest are used for the two determinations. 3. The DNA determination is based on the color reaction with indole. It has a range of 0.2–2.0 μg. DNA with a standard deviation of 0.015 μg. The average recovery of added DNA is 103 ± 4%. 4. The protein determination is a slightly modified version of the bromosulfalein method of Nayyar and Glick, with a range of 1.0–10 μg. protein and a standard deviation of 0.15 μg. The decrease in optical density relative to a reagent “blank” is proportional to the amount of protein in the sample up to a relative decrease of 60%. Factors converting the decrease in optical density to μg. protein have been established for chick embryo heart, lung, intestine, liver, and brain. 5. The specificity of both methods has been studied. It was found that a single extraction with trichloroacetic acid will remove virtually all of the small amounts of material interfering in the DNA determination.

Studies on sodium-potassium-activated adenosinetriphosphatase. VI. Its role in cation transport in the lens of cat, calf and rabbit.

Abstract The enzyme Na-K-activated ATPase, which is closely related to the active transport system of sodium and potassium in human erythrocytes, was found to occur in the epithelium of the lens of the cat, calf, and rabbit in relatively high activities. No significant activity could be detected in anterior and posterior capsule, cortex, and nucleus. Various properties of the enzyme system were determined and compared with corresponding properties of the transport system, reported by other investigators. Both the enzyme and the transport system were found to be located in the lens epithelium. They were both inhibited by ouabain, and there was good agreement between the half-maximal inhibition concentrations for the two systems in both calf and rabbit lens, when determined in media with the same K level. In the enzyme system as well as the transport system, Rb could replace K. In the rabbit lens the pH optimum of the enzyme was 7.3 as compared to 7.5 for the Rb uptake. The temperature coefficients of the two systems were 2.4 and 2, respectively. The ratios of equivalents of cation transported to moles of ATP hydrolyzed by the epithelial Na-K ATPase ranged from 2.51 to 4.15 (av. 3.07) for the three cations actively transported in calf and rabbit lens. It was concluded that the epithelial Na-K ATPase and the active cation-transport system of the lens are identical or very closely related.

STUDIES ON SODIUM-POTASSIUM-ACTIVATED ADENOSINETRIPHOSPHATASE. XI. THE SALT GLAND OF THE HERRING GULL.

Abstract The enzyme Na-K-activated adenosinetriphosphatase (Na-K ATPase) was found in high activity (2.7 moles/kg wet wt/hr) in the salt gland of the herring gull. It requires for activation both Na ( K m = 12.5 mmoles/liter) and K( K m = 1.5 mmoles/ liter), and is inhibited by the cardiac glycoside ouabain (pI 50 = 6.30). Its pH optimum is 7.2, while the Mg-activated ATPase activity has a pH optimum of 8.7. The properties of the Na-K ATPase activity suggest that it is involved in the ouabain-sensitive salt secretion by this gland. Comparison of the glandular Na-excretion rate determined by other workers with the Na-K ATPase activity per whole gland of freshly caught animals gave a ratio of 2.0 equivalents Na excreted per mole ATP hydrolyzed. This ratio falls within the range of Na/ATP ratios previously determined for several other tissues. Herring gulls maintained for 7 weeks on fresh water had only 51% of the Na-K ATPase activity per gram gland of wild animals, while the gland weight had dropped to 65%. Calculation showed that the remaining activity was just sufficient for the average daily salt intake. When four of the captive animals were given 3% NaCl to drink, all four died within 10 days, suggesting that the loss of Na-K ATPase activity could not be quickly reversed. The high molar activity of carbonic anhydrase, which was not significantly changed in the fresh water animals, indicates that this enzyme can only play a secondary nonlimiting role in the salt secretion process.

Studies of sodium-potassium activated adenosine triphosphatase: VII—inhibition by erythrophleum alkaloids

Erythrophleine and cassaine, two alkaloids occurring in the bark, leaves, and seeds of the genus Erythrophleum, were shown to be potent inhibitors of the digitalissensitive Na-K activated adenosine triphosphatase (Na-K ATPase) of rabbit brain, kidney, and ciliary body and cat choroid plexus. At 10−4 M concentration, erythrophleine inhibited Na-K ATPase activity in all four tissues completely, while cassaine at 10−4 M inhibited Na-K ATPase activity in the same tissues-an average of 86 per cent. Neither of these compounds caused significant inhibition of Mg-activated ATPase activity. n nThe inhibition curves for erythrophleine and cassaine in rabbit brain had the same general shape as the curve for ouabain. The negative logarithms of the molar inhibitor concentration at which 50 per cent inhibition of Na-K ATPase activity occurs were 6.58 for erythrophleine, 5.96 for ouabain, and 5.28 for cassaine. All three compounds caused a stimulation of approximately 6 per cent at about 1300 of their halfaximal inhibition concentrations. n nIncreasing the K concentration in the assay medium from 0 to 40 mM caused 75 per cent reversal of the inhibition of rabbit brain Na-K ATPase by 5 × 10−7 M erythrophleine as well as by 5 × 10−7 M ouabain. No reversal was obtained of the Na-K ATPase inhibition by 10−4 M erythrophleine and ouabain. n nIt is concluded that the similarity of the effects of the erythrophleum alkaloids and the digitalis glycosides on the Na-K ATPase system may explain the remarkable similarity between these two groups of compounds in pharmacological properties and in inhibitory effects on active cation transport.

Ultramicro assay of the cholinesterases

Abstract 1. 1. A method is described for the simultaneous determination of cholinesterase activity and protein content in minute quantities of tissue, e.g., 50 μg. of nervous tissue or 0.2 μl. of human serum, having activities ranging from 0.01 to 0.1 μ M AcCh/hr. 2. 2. The method for cholinesterase is based on the formation of hydroxamic acid from the remaining choline ester after incubation with the enzyme; the color developed with acid ferric chloride is measured. 3. 3. Advantages of the method are: (a) It is simple and quick, and therefore suitable for routine work; (b) enzyme activity and protein content can be determined in the same sample; (c) a variety of substrates can be employed, allowing the study of the enzyme specificity; (d) the pH of incubation can be varied from pH 5.5 to 10.0; (e) the same procedure can be used to determine choline esters in biological samples. 4. 4. The reproducibility of the choline ester determination in a range of 0.01–0.1 μ M is ±.0008 μ M and that of the cholinesterase assay is ±0.0024 μ M /hr. in a range of 0.01–0.1 μ M /hr. 5. 5. The effects of the buffer system and the preparation of the sample have been studied in some detail.

Some relations between growth and carbohydrate metabolism in tissue cultures.

Abstract 1. 1. The effects of nitrogen, oxygen and cyanide on the carbohydrate metabolism and the growth in terms of protein synthesis have been investigated in cultures of 15-day chick embryonic lung and intestine. 2. 2. On cultivation under air respiration predominates over glycolysis, but increase of the glucose level in the culture medium raises the percentage glycolysis considerably. 3. 3. On cultivation under 100 per cent N2 carbohydrate metabolism becomes completely glycolytic, while growth is almost normal. The tissues remain alive and active for the duration of the experiment (8 days). 4. 4. In cyanide (0.25–0.50 mM/1) with respiration largely inhibited, protein synthesis is severely decreased. The tissues become inactive at 4–6 days. 5. 5. On cultivation under 100 per cent O2 respiration is markedly decreased, while protein is lost from the explants. The tissues become inactive at 4 days.

Colorimetric determination of pyruvic acid and other α-keto acids in submicrogram quantities☆

Abstract 1. 1. The method of Friedemann and Haugen for pyruvic acid has been adapted to allow the determination of 0.1–1.0 μg. of pyruvic acid or other α-keto acids in samples containing at least 2 μg./ml. of these acids, with a reproducibility of 0.022 μg. (standard deviation of the observations) and a recovery of 102% (±4%). 2. 2. Deproteinization of the samples is shown to be unnecessary under the conditions specified. 3. 3. The yellow color of the original hydrazone extract in Na2CO3 solution is read at 380 mμ, omitting the addition of NaOH. This has several advantages, e.g., increased color stability, increased sensitivity, and lower blank absorption. 4. 4. A differential determination of pyruvic acid and either α-ketoglutaric or oxalacetic acid, with an average error of 2.9%, is possible on the submicrogram scale by varying the reaction time and the organic solvent.