Some kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

• 1.1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5.
• 2.2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg²⁺, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate.
• 3.3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form.
• 4.4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

     

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

• 1.1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
• 2.2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
• 3.3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. II. Effects of adenine nucleotides

• 1.1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations.
• 2.2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor.
• 3.3. Mg²⁺ decreases the inhibition produced by AMP and ADP by enhancing their I_0.5 and completely annulates the inhibitory effect of ATP.
• 4.4. In the presence of Mn²⁺ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.

     

Comparative hematology: Studies on cats including one with factor XII (hageman) deficiency

• 1.1. Cat plasma prothrombin and partial thromboplastin times are faster than human. Thromboplastin generation tests are very similar.
• 2.2. Factors VIII and V assay 24 and 13 times the human standard. Cat factors VII, X. IX, XI and XII assayed at 2.5 to 4 times human. Factors I, II and XIII fell within the human range and Fletcher was extremely low.
• 3.3. One cat lacked factor XII and showed a prolonged APTT and clotting time.
• 4.4. Cat profibrinolysin was activated by streptokinase but not by urokinase.
• 5.5. Cat platelets aggregated with the usual human aggregation agents with the exception of thrombin and ristocetin.
• 6.6. Cat erythrocytes were smaller and more numerous than human.
• 7.7. Leukocyte counts were quite variable.
• 8.8. Serum protein electrophoretic patterns differed from human in the greater migration of albumin and the presence of numerous unidentified bands.
• 9.9. Biochemical tests showed high sodium and chloride values.

     

The effect of hydrogen sulfide on the metabolism of Solemya velum and enzymes of sulfide oxidation in gill tissue

• 1.1. The addition of sulfide to sea-water in respirometer flasks stimulated oxygen uptake by intact Solemya velum; at concentrations of 0.5 and 0.8 mM, the experimental rates were 1.8 and 2.5 times control rates.
• 2.2. Extracts of gill tissue catalyzed the conversion of thiosulfate to sulfite, the production of adenosine phosphosulfate (APS) from AMP and sulfite and the formation of ATP from APS. The enzymes, thiosulfate sulfurtransferase (EC 2.8.1.1), adenylsulfate reductase (EC 1.8.99.2) and sulfate adenylyl transferase (EC 2.7.7.4) have K_m and V_max in the same range as similar enzymes in other species.
• 3.3. Calculations based on these experiments suggest that adenylylsulfate reduction is ordinarily catalyzed at no more than 8% of maximum velocity.

     

Pigment migration in fish erythrophores is controlled by alpha2-adrenoceptors

• 1.1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha₂-adrenoceptors.
• 2.2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha₂-adrenoceptors.
• 3.3. The erythrophores themselves were found to possess a K⁺-sensitive mechanism of aggregation.
• 4.4. Some similarities and differences of the alpha₂-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Combined effect of adenosine,alpha adrenergic and adenosine antagonists on serum insulin and insulin secretion from rat pancreatic islets

• 1.1. The effect of adenosine separately or in combination with alpha-1 adrenergic antagonist prazosin and alpha-2 adrenergic antagonist yohimbine as well as adenosine antagonists 8-phenyltheophylline and xanthine amine conjugate on glucose-induced insulin secretion from isolated rat pancreatic islets was studied.
• 2.2. Their in vivo effects on serum glucose and insulin levels were also investigated. Adenosine at 10 and 100 μM inhibited significantly, insulin secretion from the isolated islets whereas at 10 mM slightly increased the secretion of insulin.
• 3.3. Prazosin used at 100 μM inhibited insulin secretion. When it combined with adenosine (10 μM) it augmented the inhibitory effect of adenosine.
• 4.4. In vivo prazosin (21 mg/kg bodywt) caused a hyperglycaemia which was accompanied by hypoinsulinaemia.
• 5.5. Concurrent administration of this drug with adenosine neither affect the hyperglycaemic nor the hypoinsulinaemic effects of adenosine.
• 6.6. On the other hand, yohimbine (100 μM) has no effect neither separately nor in combination with adenosine (10 μM) in modulating the inhibitory effect of adenosine on insulin secretion.
• 7.7. When Yohimbine administered at 19.5 mg/kg body wt it did not alter serum glucose but it markedly increased the serum insulin level. Its combined administration with adenosine reduced the hyperglycaemic effect of adenosine with a remarkable increase in serum insulin.
• 8.8. Both adenosine-antagonists were ineffective in alteration of insulin secretion.
• 9.9. However, combination of 8-phenyltheophylline with adenosine (10 μM) totally blocked the inhibitory effect of adenosine on insulin secretion while xanthine amine conjugate failed to prevent this effect of adenosine.
• 10.10. These results indicate that the inhibitory effect of adenosine on insulin secretion is neither mediated via alpha-1 nor alpha-2 adrenoceptors. It might be via activation of specific adenosine receptors on rat islets which are sensitive to blockade by 8-phenyltheophylline.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

Effects of ATP and cAMP on salt and sugar (responses of chemosensilla in the blowfly)

• 1.1. The addition of cAMP to stimulating solutions of NaCl, fructose (furanose sugar), sucrose, or glucose (pyranose sugars) decreases the responsiveness of labellar chemosensilla in Phormia.
• 2.2. The addition of ATP, while decreasing the responsiveness to NaCl or fructose enhances the responsiveness to glucose and sucrose.
• 3.3. The inhibiting effect of ATP on NaCl or fructose responses is suppressed by GDPßS, an inhibitor of adenylate cyclase (and thus of cAMP synthesis); moreover GDPßS further enhances the increase in response due to ATP when added to the sucrose or glucose solutions.
• 4.4. Results suggest a possible involvement of cAMP and ATP in the taste reception mechanism in the blowfly.

     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

The effects of ATP and cAMP on the thermal tolerance of the mudpuppy,Necturus maculosus

• 1.1.|Data from previous studies suggest that cellular thermal tolerance depends in part on the availability of high-energy metabolites. To determine if a similar phenomenon is involved in the regulation of thermal tolerance in whole organisms, we treated the mudpuppy, Necturus maculosus, with drugs presumed to elevate or depress hypothalamic levels of either adenosine triphosphate (ATP) or cyclic adenosine monophosphate (cAMP).
• 2.2.|Injections, into the third ventricle, of ATP, cAMP, and theophylline (which elevates endogenous levels of cAMP) elicited dose-related increases in the critical thermal maximum (CTM).
• 3.3.|A dose-related decrease in the CTM resulted when animals were treated with 2-deoxy-D-glucose, a nonmetabolizable glucose analogue.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Purification and characterization of casein kinase II from lactating rabbit mammary gland

• 1.1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps.
• 2.2. Its K_m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K_i = 5 and 1 mM respectively).
• 3.3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration.
• 4.4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

     

Purification and properties of glycollate oxidase from Lemna minor L.

• 1.1. Glycollate oxidase has been purified to apparent homogeneity from Lemna minor L. grown on medium containing 7mM NO⁻₃.
• 2.2. The enzyme is a highly basic protein with a sub-unit molecular weight of 42,000 and a holoprotein molecular weight of 250,000.
• 3.3. The Lemna enzyme is a flavoprotein with a broad specificity for straight chain α-hydroxy acids, the preferred substrate being glycollate.
• 4.4. It is also competitively inhibited by oxalate and phenyllactate.
• 5.5. A comparison is drawn between the physical properties of glycollate oxidase from a number of higher plants and the degree of sub-unit aggregation in the resulting protomers.