Some properties of ATPase activity in the intact cells of yeast Saccharomycopsis fibuligera

• 1.1. The properties of ATPase activity were studied with the cells at the early stationary phase of Saccharomycopsis fibuligera.
• 2.2. Optimal pH for the activity was approximately 7.
• 3.3. The activity was stimulated by Mg²⁺.
• 4.4. The activity was inhibited by NaF, DCCD, oligomycin, NaN₃, NaVO₃, or PCMB but not inhibited by ouabain.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

Evidence for the presence of ion pumps in the photophores of two mesopelagic fishes: Argyropelecus and maurolicus

• 1.l. Adenosine triphosphatase (ATPase) activity has been measured on homogenates of photophores from the two mesopelagic fishes Argyropelecus and Maurolicus. This activity is equivalent for both fishes when reported to the protein content as is their O₂ consumption.
• 2.2. The activity is optimal at pH 6.8–7.5. It is not specific for ATP since GTP, ITP, UTP and CTP are also hydrolyzed to a significant extent. It is also not specific for Mg²⁺, the activity being equivalent (Argyropelecus) or higher (Maurolicus) with Ca²⁺ and high also with Co²⁺ and Mn²⁺
• 3.3. Twenty to 30 per cent of the activity measured at pH 7.4 is probably due to the mitochondrial ATPase as it is shown by oligomycin and venturicidin inhibition.
• 4.4. Activities of both fishes photophores are partly inhibited by N-N'-dicyclohexylcarbodiimide (DCCD), azide, LaCl₃, vanadate, diethylstilbestrol (DES) and N-ethylmaleimide (NEM) which are all inhibitors of ionic pumps.
• 5.5. Argyropelecus activity is sensitive to ouabaïn.
• 6.6. Our results show the presence of ionic pumps in Argyropelecus and Maurolicus photophores. If there is evidence for the absence or very low activity of a H⁺ pump, it is sure that Argyropelecus at least possess a Na⁺K⁺-ATPase.
• 7.7. The significance of a high protein content in Maurolicus photophores and of a large inorganic phosphate concentration in Argyropelecus is discussed.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations

• 1.1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg²⁺ and Mn²⁺.
• 2.2. Mg²⁺ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory.
• 3.3. Mn²⁺ produces a 10-fold rise in V_max higher than Mg²⁺. [Mn²⁺]_0.5 is much larger than [Mg²⁺]_0.5. At elevated [Mn²⁺] inhibition is observed.
• 4.4. Mg²⁺ and Mn²⁺ produce antagonistic effects on the inhibition of the enzyme by high substrate.
• 5.5. Fru-2,6-P₂ inhibits the enzyme by rising the S_0.5 and favouring a sigmoidal kinetics.
• 6.6. The inhibition by Fru-2,6-P₂ is released by Mg²⁺ and more powerfully by Mn²⁺ increasing the I_0.5.

     

Acid phosphatase in the contents of the oesophagus and stomach of the snail,Helix pomatia L.

• 1.1. In the contents of the oesophagus and stomach, one form of acid phosphatase is found. Its electrophoretic mobility is identical to that of the multiple form 3 of acid phosphatase from the hepatopancreas.
• 2.2. The enzyme is not stimulated by divalent cations. It is inhibited by molybdate, Cu²⁺, Hg²⁺. F⁻ and tartrate L+.
• 3.3. The optimum pH of the enzyme is 4.5. The K_m for paranitrophenylphosphate as substrate amounts to 0.25 mM. The enzyme is stable at a temperature of up to 55°C.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Bioelectrical potentials across the mantle epithelium of Achatina fulica

• 1.1. The shell side of the mantle of Achatina fulica is several millivolts positive to the blood side in vitro.
• 2.2. The electrical potential does not depend on Na⁺, Ca²⁺, Mg²⁺, K⁺ or HCO₃⁻ but requires the presence of chloride on the shell side.
• 3.3. The potential difference and short-circuit current ranged from 3.0 to 30.0 mV and 15.0 to 75 μA/cm² with averages at 10m V and 50 μA/cm² respectively.
• 4.4. The electrical gradient is reduced by 2,4-dinitrophenol, thiocyanate and furosemide but not by ouabain, CO₂ or acetozolamide.
• 5.5. It is suggested that the nature and mechanism of electrogenesis in Achatina parallels that of the Helix mantle.

     

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.

     

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⁺.

     

Properties of an alkaline phosphatase from the dinoflagellate Peridinium cinctum

• 1.1. Alkaline phosphatase (EC 3.1.3.1) from the dinoflagellate Peridinium cinctum, the Lake Kinneret bloom alga, has been partially purified by gel filtration.
• 2.2. The enzyme could be easily extracted using a distilled water/chloroform mixture suggesting that the alkaline phosphatase of Peridinium is particularly labile.
• 3.3. The molecular weight of the enzyme was estimated as 158,000 ± 5000. The enzyme showed a broad pH optimum (in the range pH 8.0–8.5), had a K_m of 0.45 mM for p-nitrophenylphosphate as substrate and was stable to repeated freeze/thawing cycles.
• 4.4. The enzyme was strongly activated by Mg²⁺ whereas Zn²⁺ (and to a lesser extent Cd²⁺) was an effective inhibitor of the enzyme. Cu²⁺ activated the enzyme at low concentrations, although at higher concentrations inhibited the enzyme. This effect of metals on the Peridinium alkaline phosphatase could be environmentally important since underwater hot springs, containing high concentrations of copper, enter the lake.

     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

A comparative study of the respiratory properties and physiological function of haemocyanin in two burrowing and two non-burrowing crustaceans

• 1.1. Oxygen dissociation curves were constructed for the haemolymph of two non-burrowing, Galathea strigosa and Eupagurus bernhardus, and two burrowing crustaceans, C. cassivelaunus and Nephrops norvegicus. The p₅₀ at in vivo pH values and 10°C was 12.6 Torr in G. strigosa, 23 Torr in E. bernhardus, 3.1 Torr in C. cassivelaunus and 11.5 Torr in N. norvegicus.
• 2.2. The Bohr values (Δlogp₅₀/ΔpH) were high in all species ranging between −0.96 and −1.48. Cooperativity expressed as P₅₀ averaged 3.3, 3.8 and 3.8 in G. strigosa, E. bernhardus and N. norvegicus. respectively. A lower value of 2.2 was observed in C. cassivelaunus.
• 3.3. The oxygen affinity of the haemocyanin was relatively temperature independent, the values for ΔH at pH7.9 ranging between −5.1 and −18.1 kJmol⁻¹.
• 4.4. Haemolymph respiratory gas analysis showed values similar to those previously reported in crustaceans: p_aO₂ ranging between 44 and 107 Torr and p_vO₂ values between 18 and 24 Torr.
• 5.5. Pre-/post-branchial pH differences were small in G. strigosa, E. bernhardus and N. Norvegicus, but averaged 0.09 of a pH unit in C. cassivelaunus. p_aCO₂ and P_vCO₂ values ranged between 1.4 and 2.3 Torr.
• 6.6. In buried C. cassivelaunus both pre- and post-branchial oxygen tensions decreased, as did oxygen tension overall during respiratory pauses.
• 7.7. Cardiac output values were low, ranging between 59 and 71 ml kg⁻¹ min⁻¹ for all four species and calculated stroke volumes were realistic in terms of animal size.
• 8.8. In the non-burrowing species physically dissolved oxygen accounted for 5–21% of the oxygen transported to the tissues. In the burrowing species values of 40–77% were found.

     

Effects of aluminium and pH on calcium fluxes,and effects of cadmium and manganese on calcium and sodium fluxes in brown trout (Salmo trutta L.)

• 1.1. Simultaneous measurement of calcium fluxes in brown trout, at low external [Ca] (20 μ mol 1⁻¹), provided evidence of active uptake of Ca from the medium.
• 2.2. At pH 4.5, calcium influx was inhibited and efflux was stimulated.
• 3.3. Cd and Mn, but not Al, at concentrations within the ranges found in acid waters experiencing fish population decline, inhibited calcium influx. Efflux was unaffected.
• 4.4. Cd and Mn stimulated sodium influx and efflux.

     

Nacl flux in the flounder (Pseudopleuronectes americanus) intestine: Effects of ph and transport inhibitors

• 1.1. The effects of extracellular pH on Na⁺ and Cl⁻ absorption were studied in vitro in the small intestine of the winter flounder, Pseudopleuronectes americanus.
• 2.2. Reductions in bathing solution pH inhibited J_ms^na (mucosal-to-serosal flux) and J_net^na (net flux) (r = 0.90) and J_net^Cl (r = 0.92) [due to an increase in J_sm^Cl, (serosal-to-mucosal)] and decreased short circuit current (Isc).
• 3.3. Luminal bumetanide (0.1 mM) and amiloride (1 mM) inhibited Na⁺ and Cl⁻ absorption by reducing J_ms.
• 4.4. Luminal barium (5mM) and luminal copper (100 μM) decreased J_ms^Cl and increased J_sm^Cl.
• 5.5. We conclude that reductions in extracellular pH inhibit a luminal membrane NaCl absorptive process (Na⁺-K⁺-2Cl⁻) and stimulate an electrogenic Cl⁻ secretory process.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.