Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

Summary The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu²⁺ and Fe²⁺. The rate of H₂O₂ formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na⁺ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.     

Purification and some properties of hemagglutinin from the Myxomycete,Physarum polycephalum

A new hemagglutinin was isolated from the plasmodium ofPhysarum polycephalum by salting out with ammonium sulphate followed by chromatography on DE-32, DEAE-Toyopearl and hydroxyapatite. This hemagglutinin, named physarumin, was purified 1000-fold over crude extracts. The molecular weight of physarumin was determined to be 22,000 by size exclusion chromatography on Bio-Gel P-60 and 8,700 by SDS-polyacrylamide gel electrophoresis. Physarumin agglutinated rabbit, guinea pig, horse and human erythrocytes. Physarumin-induced hemagglutination was inhibited by fetuin and ₁ glycoprotein, but not by commercially available mono-and disaccharides. Hemagglutinating activity was blocked by EDTA, and was restored by adding Ca²⁺ but not by Mg²⁺.     

Partial purification and characterization of acid phosphatase from sporulated oocysts ofEimeria tenella

Summary Acid phosphatase ofEimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and V_max values for (Peak II) were 25 mM and 1.57 mol/min/mg protein, respectively. The enzyme (Peak II) ist strongly inhibited by Hg⁺⁺, Cu⁺⁺, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Zinc increases the longevity of unfertilized sea urchin eggs

Summary We have found that Zn²⁺ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn²⁺ inhibited this proteolysis. Therefore, Zn²⁺ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.     

Inhibition ofE. coli ATPase activity by a troponin component,TN-I,and by mitochondrial ATPase inhibitor

Summary The enzymic activity of Mg²⁺-or Ca²⁺-stimulated ATPase fromEscherichia coli was inhibited by one of the troponin components, TN-I, and by mitochondrial ATPase inhibitor (F₁-inhibitor). The inhibitory ability of component TN-I against Mg²⁺-stimulated ATPase activity was lost after digestion of component TN-I with trypsin. The Mg²⁺-stimulated ATPase activity inhibited by component TN-I was completely restored by the addition of another troponin component, TN-C.     

Induction of cell contact sites by Ca2+-EDTA pulses inDictyostelium discoideum

Summary The effect of extracellular Ca²⁺ on the differentiation of the cellular slime moldD. discoideum was examined. In the case of sustained application of calcium, cells required at least 60 min for the presence of calcium to show the induction of EDTA resistant cell contact sites (csA). However, the application of 12 pulses of calcium, followed after 30 sec by EDTA, giving a total time of Ca²⁺-exposure of 6 min, could induce csA on the cell surface.     

Melezitase and maltase from the midgut ofSesamia inferens Walker (Lepidoptera: Insecta)

Summary The activity of melezitase and maltase was optimal at pH 6.2 and temperature 35–40°C and inhibited by the end-products. Melezitase activity was affected by K⁺, Li⁺⁺ andTris ions and was reduced by dialysis.Acknowledgments: The author is thankful to ProfessorR. Rakshpal for valuable guidance, and to University Grants Commission, for awarding me a Junior Research Fellowship.     

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.     

Class I and class II ribonuclease H activities inCrithidia fasciculata (protozoa)

Summary The protozoanCrithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg²⁺- and Mn²⁺- ions. However, the presence of Mn²⁺-ions is inhibitory for the Mg²⁺-dependent class II ribonuclease H activity ofCrithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

Über Struktur und Aktivität der den H2O2-Zerfall katalysierenden Cu2+-Komplexe. — VI. Differenzierung von nativer RNS und nativer DNS bzw. denaturierter DNS auf Grund der katalytischen Eigenschaften

Summary The catalysis of H₂O₂ decomposition by Cu²⁺-complexes of RNA and DNA has been investigated. It is shown that both complexes decompose H₂O₂, but only the Cu²⁺-RNA-system shows peroxidative activity too, e.g. only in this case the nucleotide bases are degraded. Thermal denaturation of DNA also leads to a Cu²⁺-complex with peroxidative activity, the latter being dependent on the degree of denaturation.

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V. Mitteilung:S. Petri, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 1778 (1966).

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12. Mitteilung überMetallionen und H ₂ O ₂; 11. Mitteilung:H. Ch. Curtius, P. Anders, R. Zell, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 2256 (1966).     

Blockade by zinc ions of K+-induced contraction and calcium in guinea pigTaenia coli

Preincubation with 0.3 mM Zn²⁺ markedly inhibited both the tonic response and Ca²⁺ binding at low affinity sites induced by K⁺ (60 mM), with smaller effects on the phasic response and the high affinity Ca²⁺ sites, inTaenia coli. However, when the muscle was kept in Zn²⁺-containing medium following the first stimulation with the K⁺, the phasic response and the high affinity Ca²⁺ sites were more severely inhibited during the second stimulation with K⁺. This probably indicates that Zn²⁺ reduced the tonic tension response to K⁺ mainly by inhibiting Ca²⁺ influx at the cell membranes ofTaenia coli. However, when Zn²⁺ is continuously present, Ca²⁺ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K⁺.     

Dissociation-constants of metal-ion-complexes with alkaline phosphatase from pig kidney

Summary Using metal-ion buffers it was possible to remove Zn²⁺, Mg²⁺ and Mn²⁺ ions of pig kidney alkaline phosphatase reversibly. The dissociation constants obtained are K_EMg:4·10^–7 M, K_EMn:4·10^–8 M and K_EZn:8·10^–13 M (22°C, pH:9.6, :0.07).Acknowledgement: The authors thank Dr.H. U. Wolf for helpful suggestions and valuable discussion and MissH. Köth for technical assistance.     

The effects of various agents in vitro on homocarnosine-carnosine synthetase from rat brain

Summary The present paper reports the first evidence that homocarnosine-carnosine synthetase from rat brain requires free sulfhydryl groups for activity. The activity of the synthetase can be stabilized by dithioerythritol and inhibited strongly by Cu²⁺, Cd²⁺, Hg²⁺, Zn²⁺, and to a lesser extent by Ca²⁺, Ni²⁺, Li, chlorpromazine, -methyl DOPA and norepinephrine at the concentrations tested.Acknowledgment. This work was supported by United States Public Health Service Grant No. NS-06137.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Malignant hyperthermia: Molecular defects in membrane permeability

Summary Malignant hyperthermia (MH), a genetically inherited disorder of skeletal muscle, is due to molecular defect in membrane permeability. The alteration in membrane permeability is suggested to be due to enhanced phospholipase A₂ activity which is responsible for the increased level in sarcoplasmic Ca²⁺. The excess Ca²⁺ is responsible for muscle hyper-rigidity and enhanced rate of glycolysis, resulting in a rapid rate of lactic acid production and a low pH in MH muscle.     

Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca²⁺-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca²⁺ influx or addition of Ca²⁺ in cellular extracts. Ca²⁺ failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca²⁺ influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K¹⁵⁸ and K¹⁸³ of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.     

Decreased rates of Ca2+-dependent heat production in slow- and fast-twitch muscles from the dystrophic (mdx) mouse

Using a newly developed microcalorimetric approach to assess the rate of energy expenditure for intracellular [Ca²⁺] homeostasis in isolated muscles at rest, we found this was lower inmdx than in control mouse muscles, by 62% and 29% in soleus and extensor digitorum longus, respectively. Differences in total and Ca²⁺-dependent rates of specific heat production betweenmdx and control were enhanced during sustained, KCl-induced stimulation of energy dissipation. These results suggest that the low sacroplasmic energy status of dystrophic muscles is not due to any excessive energy expenditure for intracellular [Ca²⁺] homeostasis.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary 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 (SO ₄ ^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°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.     

Zur Metallspezifität der Hexokinase

Summary The activity of hexokinase has been determined in the presence of different metal ions. Besides Mg²⁺, the ions Co²⁺, Ni²⁺, Mn²⁺, Zn²⁺, and Cd²⁺ show remarkable activation. The differences are explained by superposition of an activating and an inhibiting function. The specifity problem is discussed.