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.

     

Purification and some properties of apurinic/apyrimidinic dna endonucleases in rat liver

• 1.1. Three kinds of apurinic/apyrimidinic (AP) DNA endonucleases, APcI, APcII, APcIII were purified from rat liver chromatin.
• 2.2. Molecular weights of APcI, APcII and APcIII were 30,000, 42,000 and 13,000 Da, which have isoelectric points of 7.2, 6.3 and 6.2, respectively.
• 3.3. Mg²⁺ was essential for the activities of these 3 enzymes, and sulfhydryl compounds (βercaptoethanol) had a stimulatory effect on the enzyme activities while N-ethylmaleimide and HgCl₂ inhibited the enzyme activity.
• 4.4. K_m values of APcI, APcII and APcIII for AP site of DNA were 0.53, 0.27 and 0.36 μM, respectively, and AMP was the most potent inhibitor to these three enzymes among nucleotides tested.

     

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).

     

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 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.

     

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.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

Modulation of the ATP induced [Ca2+]c increase in as-30D hepatoma cells

• 1.1. The regulation of the increase in the cytosolic calcium concentration ([Ca²⁺]c) induced by extracellular ATP in AS-30D hepatoma cells was studied.
• 2.2. Homologous desensitization involving the refilling of intracellular calcium pools and the participation of protein kinase C was found.
• 3.3. Isoproterenol, forskolin and dibutyril-cyclic AMP also induced an increase in [Ca²⁺]c.
• 4.4. Interestingly, synergism was found for isoproterenol or forskolin and ATP.
• 5.5. The results suggest that there are two pathways for mobilizing [Ca²⁺] in AS-30D hepatoma cells; one is activated by ATP receptors and the other by cyclic AMP.

     

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⁺.

     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

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⁻.

     

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.

     

Synaptic transmission at high pressure: Effects of [Ca2+]o

• 1.1. The effects of pressure on synaptic currents were examined in crayfish abdominal muscles.
• 2.2. Helium pressure (10.1 MPa) considerably decreased extracellulariy-recorded excitatory junctional potentials associated with increased short-term facilitation.
• 3.3. These effects could be mimicked by a reduction of [Ca²⁺]_o, and partially compensated by an increase in [Ca²⁺]_o.
• 4.4. Pressure also reduced the amplitude of the extracellular nerve terminal potentials (ENTP) by up to 25%, and significantly increased synaptic delay in a [Ca²⁺]_o-dependent manner.
• 5.5. The interaction between compression and various [Ca²⁺]_o were analysed in terms of an existing model of transmitter release. The results were consistent with the hypothesis that high pressure decreases the maximal Ca²⁺ influx into nerve terminals.
• 6.6. The decreased ENTP and increased synaptic delay suggest that additional processes may be involved in pressure effects on synaptic transmission.

     

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.

     

High energy organophosphate levels in the myocardia of cold acclimated and cold-hypoxic freshwater turtles,Chrysemys picta

• 1.1. Hearts of active, cold-acclimated, and cold-acclimated hypoxic turtles were assayed for their content of ATP, ADP, AMP and phosphocreatine.
• 2.2. Energy levels showed a significant discharge during the first 2 weeks of cold and cold-hypoxia, but remained relatively stable during an additional 2 weeks of cold exposure.
• 3.3. The functional importance of these changes is discussed.

     

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.

     

The effect on creatine kinase release of altered conditions in the Ca2+ paradox

• 1.1. Release of creatine kinase (CK) in the Ca²⁺ paradox of the Langendorff-perfused rat heart is dependent on the conditions of Ca²⁺ depletion and Ca²⁺ repletion.
• 2.2. CK release is reduced by raising [Ca²⁺]_o during Ca²⁺ depletion and progressively increased by extending the Ca²⁺ free period from 2 to 5 min.
• 3.3. CK release is reduced by decreasing the electrochemical gradient for Ca²⁺ during Ca²⁺ repletion.
• 4.4. The findings are discussed in the light of current hypotheses for the biochemical mechanisms that underlie the Ca²⁺ paradox.

     

Modulation of gastric mucosal calcium channel activity by mucus glycoprotein

• 1.1. The effect of gastric mucus glycoprotein on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The ⁴⁵Ca²⁺ uptake into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact mucus glycoprotein, was found significantly inhibited (59%) by the acidic glycoprotein fraction. This effect was associated with the sialic acid and sulfate ester groups of the glycoprotein, as their removal caused a loss in the inhibition.
• 2.2. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels showed a 50% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 3.3. The channel protein phosphorylation was inhibited by the acidic mucus glycoprotein, which also interfered with the binding of EGF to the channel protein. The inhibitory effect was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following their removal.
• 4.4. The results suggest that the acidic gastric mucus glycoproteins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Regulation of buccal mucosal calcium channel activity by salivary mucins

• 1.1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of ⁴⁵Ca²⁺while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively.
• 2.2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation.
• 3.3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 4.4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups.
• 5.5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.