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).

     

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.

     

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.

     

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.

     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

Properties and distribution of Mg2+-HCO−3-ATPase in brush border membranes isolated from rat small intestine

• 1.1. The small intestine was cut into seven segments and properties and distribution of brush border Mg²⁺-HCO⁻₃-ATPase activity in each segment were examined.
• 2.2. The optimal Mg²⁺ concentration was 1.0 mM.
• 3.3. The optimal HCO⁻₃ concentration was 100 mM in the first (duodenal), 50 mM in the 3rd and 40 mM in the 5th segment, respectively.
• 4.4. The optimal pH value was about 9.0.
• 5.5. l-phenylalanine (above 1 mM) and SCN⁻ (above 50 mM) significantly inhibited both Mg²⁺- and Mg²⁺-HCO⁻₃-ATPase activity.
• 6.6. The enzyme activity was found to be highest in the duodenal segment and then gradually decreased in consecutive segments as well as β-glycerophosphatase, Na⁺-K⁺-ATPase and supernatant carbonic anhydrase.
• 7.7. The functional significance of this ATPase and the relationship with carbonic anhydrase was discussed.

     

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.

     

Thermostability and activation by divalent cations of the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum

• 1.1. The activation energy of the membrane bound H⁺-pyrophosphatase is 44.9 k J·mol⁻¹, for the detergent solubilized enzyme is 55.9 kJ·mol⁻¹.
• 2.2. The Arrhenius plots obtained for pyrophosphatases of Rhodospirillum rubrum show no breaks.
• 3.3. At 70°C, the membrane-bound pyrophosphatase is more stable in the presence of either Mg²⁺ or Zn²⁺ than in their absence.
• 4.4. At 65°C, an activator effect of Mg²⁺ or Zn²⁺ was observed. Nevertheless, at 70°C no activation was obtained.
• 5.5. The activator effects of Mg²⁺ or Zn²⁺ were depended of their concentration.

     

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.

     

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⁺.

     

The enzymatic properties of smooth muscle myosin B from dog colon

• 1.1. Smooth myosin B and myosin A were prepared from dog colon and their enzymatic properties were studied.
• 2.2. Colonic myosin B with two light chain corresponding to L₂ and L₃ in skeletal myosin showed much lower ATPase activities than rabbit skeletal myosin B.
• 3.3. The Mg²⁺-ATPase of myosin B was activated at high magnesium concentrations with the maximum activation between 10⁻³ and 10⁻²M and showed only a slight dependence on KCl concentration. On the other hand, Mg²⁺-ATPase activity of myosin A decreased with decreasing KCI concentration, suggesting the activation by actin of colonic myosin ATPase as much as skeletal myosin ATPase.
• 4.4. The pH dependence of Ca²⁺-ATPase showed a U-shaped curve although above pH 8.5 the activity was suppressed rapidly. The activity-ionic strength curve indicated that Ca²⁺- and ethylenediamine-tetraacetic acid (EDTA)-ATPase activities increased with increasing KCI concentration.
• 5.5. Mg²⁺-ATPase was fairly stable to urea treatment, whereas EDTA- and Ca²⁺-ATPase were activated by a low concentration of urea, followed by an inhibition.
• 6.6. These results were discussed as compared with those of skeletal myosin B.

     

Partial purification and characterization of intestinal alkaline phosphatase of the rainbow lizard Agama agama

• 1.1. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase EC 3.1.3.1.) was extracted from the small intestines of the rainbow lizard Agama agama, partially purified by DEAE-cellulose and Sephadex G-200 column chromatography and characterized.
• 2.2. The enzyme had an optimum pH at 9.5 in sodium carbonate/bicarbonate buffer: a K_m of 1.6 mM with p-nitrophenyl phosphate; a molecular weight of 132,000; was inhibited by Zn²⁺, EDTA, urea and phenylalanine; stimulated by Co²⁺, Mn²⁺ and Mg²⁺, but Ca²⁺ had little or no effect on the activity of the enzyme.
• 3.3. The inhibition by urea was non-competitive, that by phenylalanine was uncompetitive. The enzyme was heat-labile.

     

A brief re-examination of the function and regulation of extracellular magnesium and its relationship to activity in crustacean arthropods

• 1.1. The magnesium ion [Mg²⁺] plays an important role as a co-factor in enzyme systems and as a modulator of the haemocyanin of crustacean arthropods.
• 2.2. Mg²⁺ is actively regulated in most decapod crustaceans via the antennal gland. The degree of regulation can be correlated to some extent with the “activity” of a particular species although there are “exceptions to the rule”.
• 3.3. Intraspecific studies indicate that there is a clear relationship between haemolymph [Mg²⁺] and the level of activity in particular crustacean species.
• 4.4. A plea is made for the investigation of temporal changes in the [Mg²⁺] of the haemolymph of a number of crustaceans and for more studies of Mg²⁺ homoeostasis in general.

     

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.

     

Renal response to hypoxia in carp,Cyprinus carpio: Changes in glomerular filtration rate,urine and blood properties and plasma catecholamines of carp exposed to hypoxic conditions

• 1.1. Changes in glomerular nitration rate (GFR), urine and blood properties and plasma catecholamines of carp were investigated during and following hypoxia.
• 2.2. GFR and urine flow decreased with increased urinary concentrations of bio-components, except protein, in the course of hypoxia.
• 3.3. Decreases in blood pH, and increases in haematocrit value and plasma K⁺, Ca²⁺, Mg²⁺, inorganic phosphate (P_i), ammonia, lactic acid and catecholamines (CAs) were observed as hypoxia progressed.
• 4.4. Increased GFR and urine flow, and higher values for urinary components, except protein, compared with those of the control were found in the initial post-stress stage.
• 5.5. The possible significance of increased plasma CAs in relation to changes in renal function in hypoxic carp is discussed.

     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.

     

On the role of fumarate reductase in anaerobic carbohydrate catabolism of Mytilus edulis L

• 1.1. The role of the fumarate:NADH oxidoreduction in the anaerobic glycolysis of the sea mussel is examined and discussed.
• 2.2. Fumarate reductase activity is present in submitochondrial particles especially from adductor muscle, digestive gland and mantle.
• 3.3. The pH optimum of the enzyme complex is 7.9; the approx K_m's for NADH and fumarate are 4.0 × 10⁻⁵ M and 6.3 × 10⁻⁵ M, respectively.
• 4.4. The enzyme complex is inhibitied by amytal, antimycin, ethanol, malonate, phosphate, rotenone, and succinate, and stimulated by Mg²⁺.
• 5.5. It is concluded that part of the mitochondrial respiratory chain is involved in the reduction of fumarate by NADH, comprising site 1 of the oxidative phosphorylation.

     

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⁻.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

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.