Alkaline phosphatase from Basidiobolus haptosporosus: Comparison with alkaline phosphatases from rainbow lizard and man

• 1.1. DEAE-cellulose chromatography of mycelial alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from Basidiobolus haptosporosus, produced three iso-enzymes “A”, “B” and “C”.
• 2.2. Fraction “A” was further characterized and showed maximum activity at pH 10 in 0.1 M sodium carbonate-bicarbonate buffer.
• 3.3. The enzyme was stimulated by Mg²⁺, Co²⁺ and Mn²⁺ and inactivated by Zn²⁺, Cu²⁺, EDTA, citrate and tartrate.
• 4.4. Phosphate ions inhibited it competitively, phenylalanine uncompetitively and urea noncompetitively.
• 5.5. It was heat stable for 60 min at 37°C but labile above 55°C.
• 6.6. Its K_m with p-nitrophenylphosphate was 0.5 mM; its estimated molecular weight was 160,000.
• 7.7. The results are compared with the properties of alkaline phosphatases from the rainbow lizard and man and discussed in terms of a triadic association between the fungus, the lizard and man.

     

Phospholipase A activity of culture supernatants from Streptococcus mutans strain 6715

• 1.1. Phospholipase A activity was found in the culture broth of growing cultures of Streptococcus mutans strain 6715.
• 2.2. The amount of enzyme activity was proportional to the cell density of the cultures.
• 3.3. The enzyme had a pH optimum of 7.0 and was inactivated at temperatures greater than 45°C.
• 4.4. The enzyme was Ca²⁺-dependent, since both EDTA and EGTA were inhibitory and Ca²⁺ was stimulatory.
• 5.5. Analysis of the fatty acid products resulting from the enzyme's action on 1-palmitoyl-2-oleoyl phosphatidylcholine indicated the enzyme to be a phospholipase A₁, (EC 3.1.1.32).

     

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.

     

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.

     

Inhibition kinetics of brain butyrylcholinesterase by Cd2+ and Zn2+, Ca2+ or Mg2+ reactivates the inhibited enzyme

• 1.1. The inhibition kinetics of sheep brain butyrylcholinesterase (BChE) (acylcholine acylhydrolase, EC 3.1.1.8) by Cd²⁺ and Zn²⁺ has been studied.
• 2.2. K_s has been determined as 0.14mM. Cd²⁺ and Zn²⁺ were the hyperbolic mixed-type inhibitors of BChE. Ca²⁺ and Mg²⁺ had no effect on the enzyme activity in the experimental conditions.
• 3.3. But when the enzyme was inhibited by 0.1 mM Cd²⁺ or Zn²⁺, Ca²⁺ and Mg²⁺ reactivated the inhibited form of BChE.

     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

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.

     

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⁻.

     

Evidence that the alkaline P-nitrophenylphosphate phosphatase from Halobacterium halobium is a manganese-containing enzyme

• 1.1. Alkaline p-nitrophenylphosphate phosphatase of Halobacterium halobiium, either purified or in crude extracts, was progressively inactivated by treatment with several metal chelators.
• 2.2. The activity of treated crude extracts was fully restored in the presence of 25–50 μM Mn²⁺ or 1 mM Co²⁺, and partially restored in the presence of 1 mM Cd²⁺.
• 3.3. Zn²⁺ ions, as well as other divalent cations tested, were without effect.
• 4.4. In the presence of a saturating concentration of Mn²⁺, but not Co²⁺ or Cd²⁺, the activity of the metal-depleted enzyme reached values well over the native control activity.
• 5.5. Activation of the metal-depleted enzyme by Mn²⁺ showed cooperative kinetics, whereas activation by Co²⁺ showed Lineweaver-Burk kinetics.
• 6.6. The results suggest that the enzyme contains two different types of metal-binding sites: essential site(s), occupied by endogenous Mn²⁺ ions, and regulatory site(s), that can be occupied by exogenous Mn²⁺ with an activating effect.

     

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

• 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
• 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn²⁺, Zn²⁺ and Mg²⁺. The K_m for Mg²⁺ is lower than the physiological concentration of cation in the tissue, whereas its K_m for Mn²⁺ and Zn²⁺ is greater than the respective in vivo concentrations.
• 3.3. The joint action of Mg²⁺ and Zn²⁺ increases the affinity of the enzyme for the substrate Fru-1,6-P₂, though V_max is reduced.
• 4.4. Na⁺ strongly inhibits the enzyme even at very low concentrations. K⁺ has no effect whatsoever.

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

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.

     

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.

     

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.

     

Hen's egg yolk alkaline phosphatase: General characterization and kinetic stuy with inhibitors

• 1.1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn²⁺?) composed of two identical inactive subunits.
• 2.2. A second metal site preferably binds Mg²⁺ (15-fold activation). Me(II))H₂O)H⁺, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion.
• 3.3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit.
• 4.4. Uncompetitive inhibition with L-phenylalanine and analogues suggests a phosphorylated intermediate.
• 5.5. Inhibition is weakly competitive with P_i strong non-competitive with PPi as compared to Mg²⁺-free PP_i, and partially competitive with arsenate.
• 6.6. The purified enzyme is stabilized and activated by amines and proteins.

     

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).

     

Calcium-dependent potassium conductance in the photoresponse of a nudibranch mollusk

• 1.1. The response to light of Hermissenda photoreceptors when recorded intracellularly without interference from synaptic and action potentials consisted of three phases: an early depolarization (ED) followed by hyperpolarization (dip) and subsequent depolarization (tail).
• 2.2. The ED and the dip were associated with increased membrane conductance while decreased membrane conductance was involved with the tail.
• 3.3. The dip reversal potential was − 82.1 ± 5.3 mV and its amplitude varied inversely with the log of [K⁺].
• 4.4. Perfusing with agents which block K⁺ current like 4AP, Quinine, Quinidine or injection of TEA eliminated the dip and its associated increased membrane conductance, thus further supporting the role of K⁺ conductance in producing the dip.
• 5.5. The dip was enhanced by increased [Ca²⁺]_o, reduced by decreased [Ca²⁺]_o and abolished together with its associated increased membrane conductance when perfused with either D600, Cd²⁺, Mg²⁺, Mn²⁺, or Co²⁺, which block transmembrane Ca²⁺ current.
• 6.6. The dip and its associated increased membrane conductance were abolished by intracellular injection of EGTA and enhanced by perfusion with Ruthenium red.
• 7.7. Intracellular injection of Ca²⁺ mimicked the dip: membrane conductance was increased and the cell hyperpolarized.
• 8.8. These results indicate that the increase in intracellular [Ca²⁺] is primarily responsible for the light-induced increase of K⁺ conductance during the dip. The possible source of the Ca²⁺ is, at least in part, extracellular due to activation of an inward Ca²⁺ current.

     

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.

     

Kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis

• 1.1. The kinetic and regulatory properties of phenylalanine ammonia-lyase from Citrus sinensis fruit tissue were investigated. The substrate specificity of the enzyme was determined as well as the effects of pH and temperature on the catalytic activity.
• 2.2. The enzyme exhibits negative homotropic effects between the substrate binding centra.
• 3.3. Binding of l-phenylalanine to the enzyme is characterized by two K_m-values; K_m^L = 13 μM and K_m^H = 52 μM; with a Hill-interaction coefficient of 0.75.
• 4.4. The enzyme is subject to product inhibition by trans-cinnamate, but the effects of allosteric effectors and inhibitors seem to be of much greater importance in the short-term regulation of phenylpropanoid metabolism in Citrus sinensis.
• 5.5. The enzyme activity was found to be modulated by end-products of diverging metabolic pathways, viz. umbelliferone, scopoletin, naringenin, quercetin, kaempferol, benzoic acid and gallic acid.