Unfolding and release of heme from human hemoglobins A,S and F

• 1.1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants.
• 2.2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F.
• 3.3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions.
• 4.4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 μM the anionic detergent sodium dodecyl sulfate.
• 5.5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

     

Serotonin,histamine and somatostatin modulation of PMA-stimulated phosphorylation of 130kDa Ca2+ pump-like protein from rat cerebellum synaptosomal membranes

• 1.1. Influence of some neurotransmitters and neuromodulators on the PMA-stimulated phosphorylation in vitro of calcium pump-like protein from rat cerebellum synaptosomal membranes was examined.
• 2.2. The prolonged time (up to 6 min) of synaptosomal membranes preincubation with 1 and 10 μM serotonin results in the increase of phosphorylation. The decrease of phosphorylation up to 80% of control value was observed for 100 μM serotonin.
• 3.3. The most stimulating effect on 130kDa protein phosphorylation was observed with 1μM of histamine (160% of control value).
• 4.4. 1 and 0.1 μM somatostatin triggered a short-time transient increase of 130 kDa phosphorylation (up to 135% of control value).

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

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).

     

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.

     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.

     

Effect of berenil on polyamine metabolism in primary cultured rat hepatocytes

• 1.1. Putrescine and spermidine content increased in hepatocytes during culture. In the presence of 10 μM Berenil, putrescine content was further increased, while the increase of spermidine was prevented.
• 2.2. Ornithine decarboxylase activity was markedly reduced, and to a lesser extent also S-adenosyl-methionine decarboxylase activity.
• 3.3. Berenil appears to promote an increase in the transformation of spermidine into putrescine, and to inhibit the polyamine efflux.

     

Plasma lipid transport in the horse (Equus caballus)

• 1.1. Equine plasma contains lipoproteins corresponding to very low density (VLDL), low density (LDL) and high density lipoproteins (HDL).
• 2.2. HDL accounts for approximately 60% of plasma lipoprotein mass and consists of a single population of particles.
• 3.3. LDL is heterogeneous comprising three discrete subfractions.
• 4.4. Two proteins are found in the region of apolipoprotein (apo) B-100 in VLDL and LDL and a third similar to apo B-48 is in VLDL.
• 5.5. Lecithin:cholesterol acyl transferase is active in plasma and hepatic lipase and lipoprotein lipase are evident in post-heparin plasma.
• 6.6. There is no significant cholesteryl ester transfer protein activity.

     

Effect of polyunsaturated fatty acids on the growth of fish cells in culture

• 1.1. The effects on growth of supplementing the medium with (n-3) and (n-6) polyunsaturated fatty acids (PUFA) were investigated in Atlantic salmon (AS) and turbot (TF) cell lines.
• 2.2. Neither cell line grew in the absence of serum, and addition of increasing percentages of serum resulted in graded increases in cell growth in both cell lines.
• 3.3. The growth of AS cells was stimulated by supplementing the medium with both (n-6)PUFA and (n-3)PUFA at 5–25 μM, especially 18:3(n-3) and 20:5(n-3).
• 4.4. Intermediate concentrations (15–20 μM) of 18:2(n-6) and 18:3(n-3) increased cell growth in TF cells, although only after 8 days in culture.
• 5.5. In contrast, both (n-3) and (n-6)PUFA at 25 μM tended to inhibit the growth of TF cells, and in longer incubations caused cell death.
• 6.6. The inhibition of TF cell growth rate and, in particular, the cell death induced by 25 μM PUFA could be abolished by the addition of vitamin E to the medium.

     

Ionic dependence and vanadate sensitivity of (Na+, K+)-ATPase isolated from branchial sac of ascidians

• 1.1. Ion dependence and vanadium-induced inhibition on branchial sac ATPase in five species of ascidian Phlebobranchiata (vanadium-accumulating) and Stolidobranchiata (iron-accumulating) were studied.
• 2.2. The ATPase was obtained from the microsomal fraction, which was prepared from each ascidian branchial sac.
• 3.3. The ATPase was dependent on Mg²⁺ and activated by exogenous Na⁺ + K⁺.
• 4.4. Ouabain inhibited the ATPase activity in vitro, 10 μM to 100 μM vanadate, in vitro, suppressed the (Na⁺, K⁺)-ATPase.

     

Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Kinetic studies of catechol-o-methyltransferase from the brain of the African catfish,Clarias gariepinus

• 1.1. Optimum in vitro conditions, and kinetics of the enzyme catechol-O-methyltransferase from the brain of the male African catfish were studied.
• 2.2. A saturated level for S-adenosylmethionine, as methyldonor, and magnesium as cofactor was reached at 5 μM and 10 mM, respectively.
• 3.3. The addition of ascorbic acid, as an antioxidant, and tranylcypromine, as a MAO inhibitor, was not necessary, during incubations with fore-brain homogenates.
• 4.4. Kinetic analysis of the methylation of catecholestrone, catecholestradiol and dopamine showed K_m values of 1.2, 0.6 and 0.5 μM, respectively.
• 5.5. The affinity of the catecholsubstrates for the enzyme catechol-O-methyltransferase is much higher in the brain of the African catfish than in tissues of mammals.

     

Isolation of crustacean egg yolk lipoproteins by differential density gradient ultracentrifugation

• 1.1. Egg yolk lipoproteins from four species of Crustacea were isolated by differential density gradient ultracentrifugation.
• 2.2. Egg yolk proteins from freshwater prawn, striped stone crab and mitten crab consissted of high-density lipoprotein (HDL) and lipid-free protein, while low-density lipoprotein (LDL) was present in the egg yolk protein of sand crayfish as well as HDL and lipid-free protein.
• 3.3. HDL was a major component in the egg yolk proteins from four species of Crustacea. HDL was identical to egg yolk lipovitellin.
• 4.4. Both HDL and LDL possessed phospholipid as a major lipid.
• 5.5. HDL, but not LDL, contained carotenoids. The color of HDL from mitten crab showed a reddish purple and was distinct from other Crustacea whose color was orange. The reddish purple color was characterized by an absorption flexion at 600–650 nm.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Selective effect of melatonin on the proliferation of lymphoid cells

• 1.1. The present study was designed to investigate the effect of melatonin on the proliferation of normal lymphocytes and certain T-lymphomas and myelomas under in vitro conditions.
• 2.2. The results revealed that administration of 200 μM melatonin inhibited significantly the incorporation of [³H]thymidine into both normal mouse and human lymphocytes and T-lymphoblastoid cell lines.
• 3.3. On the contrary, melatonin provoked an increase of myeloma cell proliferation.
• 4.4. The influence of melatonin on hybridoma cell lines was negligible.
• 5.5. Collectively, these data demonstrated that the chief pineal indole affect selectively the processes of lymphoblastoid cell growth.

     

Comparison of the intravascular metabolism of cholesteryl esters and apoproteins of plasma low- and high-density lipoproteins in the rat (Rattus norvegicus), an animal species without plasma cholesteryl ester transfer activity

• 1.1. The intravascular metabolism of the cholesteryl esters (CE) and apoproteins of low density lipoproteins (LDL) and high density lipoproteins (HDL) was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA).
• 2.2. The apoproteins and the CE of LDL had identical catabolic rates, and there was no transfer of LDL CE to other lipoprotein classes.
• 3.3. The CE of the HDL, however, had higher catabolic rates than the apoproteins, and there was transfer of HDL CE to LDL but not to very low density lipoproteins.

     

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.

     

Prostaglandin E1 and analogs of prostacyclin influencing superoxide production by the human neutrophil nadph oxidase system

• 1.1. The effects of prostaglandin (PG) E₁, and I₂ analogs (OP-41483 and OP-2507) on the Superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated.
• 2.2. In a whole-cell system, OP-2507 inhibited the Superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action.
• 3.3. The concentration of the drug required for 50% inhibition of the oxidase (IC₅₀) was 21 μM.
• 4.4. In a cell-free system, however, the drug in concentrations of < 100 μM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent.
• 5.5. Although PGE₁ and OP-41483 did not inhibit the Superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC₅₀ values of 44 and 170 μM, respectively.
• 6.6. In addition, in the cell-free system, the K_m value for NADPH of the oxidase was unchanged by PGE₁.
• 7.7. The results suggest that the PGI₂ analog, OP-2507, is a possible superoxide-scavenger and that PGE₁ inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

     

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.