Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Limited proteolysis of lactate dehydrogenase from procine heart with trypsin: Characterization and reactivation of the fragments

Summary The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD⁺ and sulfite, was digested with trypsin over a period of 12–16 h³. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states.The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80–90% of the catalytic activity. It could be demonstrated that the behaviour of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behaviour of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD–SO₃ ^– were associated to give tetramers.The reaction of antibodies against native LDH with the dimers reactivated with NAD–SO ₃ ^– demonstrated that the native LDH and the dimers have the same surface determinants.     

Conformational flexibility of poly (dG-m5dC) under very low salt conditions

Summary The methylated DNA polymer poly (dG-m⁵dC) which exhibits a B helical conformation in solutions containing 20 mM NaCl, undergoes a gradual and reversible transition to the Z conformation as the NaCl concentration is lowered. The midpoint of this transition occurs around 5–6 mM NaCl. The conformational flexibility of this polymer at such low NaCl concentrations opens up the possibility of studying the effects of other perturbants with negligible interference from salt concentration effects.     

Ion-solvent interaction: Viscosity,apparent molar volume and conductivity of univalent electrolytes in dioxanewater mixtures at different temperatures

Summary The viscosity, apparent molar volume and conductivity of NaCl, KCl, NaBr, KBr, NaNO₃ and KNO₃ solutions at mass fraction of dioxane (10, 20 and 30%)-water mixtures at 30–45°C±0.01°C have been measured. The ions appear to interact appreciably, and the ion-solvent interaction is of the order NO₃ ^–>Br^–>Cl^–.     

Large scale isoelectric focusing analysis of the non-histone proteins of the nucleus: Isolation of components with alkaline isoelectric points

Summary After large scale isoelectric focusing of rat liver non-histone protein in polyacrylamide gel, pH range 4–8.6, the only protein material found outside the gradient was present in the cathode solution (20 mM NaOH). This was low mol. wt protein material (approximately 10,000) with an acidic amino acid composition. It bound 5–6 times its own weight of basic ampholine carrier ampholytes to give a complex with a pI of 8.82. This could be dissociated by dialysis against 1 M NaCl.We are indebted to the Yorkshire Cancer Research Campaign for financial support for this project.     

Über die Molekulargrösse des Hämovanadins

Summary The red-brown haemovanadin which is contained in the haemolysate (Henze Solution) of blood cells of the ascidiaPhallusia mamillata Cuvier is a chromoproteid with trivalent vanadium as the central atom and sulphuric acid bound coordinatively. It is found to have a molecular weight of 24,400±1,900 according to the estimation of the diffusion coefficient (D_5°=6.87±0.2 · 10^–7 cm² · s^–1 at pH 2.5–2.8).

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Dem Entdecker des Hämovanadins zum 80. Geburtstag gewidmet.     

Über die Beeinflussung der Ca-Aufnahme in Lipidextrakte aus Mikrosomen und Mitochondrien des Herzens durch Digitoxin

Summary Lipids were isolated by chloroform-methanol extraction from mitochondrial and microsomal fractions of guinea-pig hearts. In the presence of digitoxin (10^–9-10^–6 g/ml) 15–30% more radioactive Ca was taken up by the lipid extracts than under control conditions, but the total amount of Ca in this phase remained unchanged. Thus, digitoxin produced an increase in the specific activity of the lipid-bound Ca which may be explained by an increased exchangeability of this Ca fraction. This effect of digitoxin might result in an improved availability of the lipid-bound Ca for Ca-dependent functions (e.g. contraction) of the heart muscle cell.     

Soybean photosynthesis: simultaneous14CO2 and O2 estimates

Summary A method of estimating the photosynthetic rate of soybean leaves using an oxygen electrode is presented. The procedure is rapid, requires small samples and compares favourably with estimates by other techniques. Light saturation occurs at 1200 E·m^–2·sec^–1. The apparent K_m for HCO ₃ ^– is 3.2 mM at pH 7.6.     

Net ATP synthesis by running the red cell calcium pump backwards

Summary Ca²⁺ loaded inside-out vesicles from human red blood cells, yielding Ca²⁺ into a Ca²⁺ free medium with 4 mM EGTA, 2 mM ADP and 10 mM phosphate, produced an excess of 14.9 pmoles · min^–1 · (mg protein)^–1 of ATP compared to controls in which the transmembrane Ca²⁺ gradient was abolished by the ionophore A 23 187.We are obliged to Dr H. Fey and Miss H. Pfister (Veterinarybacteriological Institute Bern) and Dr H. Porzig (Pharmacological Institute Bern) for help and advice.     

Caclium causes the biphasic dose-response curve for pancreatic amylase secretion

Summary High concentrations of bethanechol (10^–4 to 10^–3 M) were effective stimulants of amylase secretion from the mouse pancreas if incubations are performed in low [ca²⁺] (0.1 mM) solutions but not if normal Krebs solution (2.56 mM Ca²⁺) was used. This inhibitory effect of Ca²⁺ at high secretagogue concentrations did not appear to be mediated through the microtubules or microfilaments.     

Distribution of chromium in red kidney beans (Phaseolus vulgaris L.)

Summary Distribution of both Cr³⁺ and CrO₄ ^2– in bean shoots followed a markedly acropetal gradient. Chemical fractionation of radiochromium accumulated in the edible bean pods indicated the greatest association (70–75%) with ionic forms (extractable by weak mineral acids).     

Adenosine uptake in pyrimidine 5′-nucleotidase deficient human erythrocytes via a high affinity transport system

Summary Using a pulse-labeling technique,¹⁴C-adenosine uptake into pyrimidine 5-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed K_m values of 2.0×10^–3 mM and 0.2 ×10^–3 mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.     

Calcium and the contractile response to prostaglandin in the smooth muscle of guinea-pig stomach

Summary In the longitudinal smooth muscle of guinea-pig stomach, verapamil (10^–5 M) which showed marked suppression of high K-induced contractures, did not suppress the contractile response to PGE₁ (1.5×10^–9 to 10^–6 M) markedly. These results suggest that the contractile mechanism of PGE₁ in guinea-pig stomach may mainly depend on a release of bound Ca in the cell and partly depend on a Ca influx from the extracellular origin.     

Trypsin activity in the hepatopancreas ofMacrobrachium lamarrei (Crustacea: Decapoda)

Summary Trypsin from the hepatopancreas ofMacrobrachium lamarrei showed optimum activity at pH 7.5 and temperature 45°C. The enzyme activity increased with the increase in incubation period and enzyme concentration. Michaelis constant of the enzyme was 2.38×10^–2 M.Acknowledgments. The authors are thankful to University Grants Commission, India for the award of a Junior Research Fellowship.     

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex

Summary Fast and very slow hydrolyses of des-Arg⁹-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, K_m values, for des-Arg⁹-bradykinin and bradykinin were found to be 0.24 mM and 4.4 M, and the maximum velocities, V_max values (mol·min^–1·mg protein^–1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.Acknowledgments. We thank Dr Kinzo Nagasawa of Kitasato University for encouragement in this work and Dr Ervin G. Erdös, University of Texas, Health Science Center, Dallas, for valuable discussion and suggestions.Abbreviations used: Z, carbobenzoxy; Bz, benzoyl; ACE, angiotensin I-converting enzyme; BK, bradykinin; HPLC, high performance liquid chromatography.     

pH-dependent action of NaCl on isolated guinea pig's ileum

Riassunto L'NaCl, aggiunto a un bagno — in cui è sospeso un frammento di ileo di cavia — sino a concentrazioni finali comprese fra 2·10^–3 e 10^–2, agisce come contratturante a pH=8,0 e come antagonista competitivo dell'acetilcolina e dell'istamina a pH=5,3.     

Effects of various inhibitors and 2,4-dinitrophenol on adenosine triphosphatase from Malpighian tubules ofLocusta migratoria L

Summary Both Mg²⁺-ATPase and HCO ₃ ^– -stimulated ATPase activity were inhibited by sodium azide and to a lesser extent ethacrynic acid and amiloride. 1 mM DNP stimulated Mg²⁺-ATPase activity by 22% and HCO ₃ ^– -stimulated ATPase activity by 7%.     

Ion-solvent interaction from viscosity,apparent molar volume and conductivity data of bromates,iodates and sulphates of potassium and sodium in dioxane-water mixtures at different temperatures

Summary The viscosity, apparent molar volume and conductivity of KBrO₃, NaBrO₃, KIO₃, NaIO₃, K₂SO₄ and Na₂SO₄ at mass fraction of dioxane (10, 20 and 30%) — water mixtures at 30–45°C±0.01°C have been measured. The ions appear to interact and the ion-solvent interaction is of the order BrO ₃ ^– >IO ₃ ^– >SO ₄ ^2– .     

Inhibition of glucose transport in human erythrocytes by 2,3-dioxoindole (Isatin)

10 mM isatin (2,3-dioxoindole) inhibited glucose influx into human erythrocytes by over 30%. The inhibition is of the competitive type, where the affinity constant (K_t) was increased from 5.71 (control) to 11.11 mM in the presence of isatin with no change in V_max (130 nmol/min/ml packed cells). The observed inhibition of sugar transport by isatin was not mediated through membrane–SH groups accessible to iodoacetate, iodoacetamide, DTNB, DNP or sodium arsenite. Isatin inhibited sugar transport in the presence of 2 mM harmaline, an alkaloid inhibitor of Na⁺, K⁺–ATP_ase activity. The inhibition was non additive which suggests that these two compounds interact with the same or a similar site on the erythrocyte membrane.     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.