Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C.

Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents — octyl-β-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in Vmax (35%) without a significant change in Km. Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.

Protective effect of some amino acids synthesized derivatives and their chelates on Escherichia coli under X-ray irradiation.

The protective effects of novel synthesized derivatives of some amino acids — nicotinyl-L-tyrosinate and nicotinyl-L-tryptophanate schiff bases and their Cu(II) and Mn(II) chelates on growth, survival and membrane-associated ATPase activity of E. coli under X-ray irradiation were investigated. The specific growth rate and survival of E. coli were decreased at 10, 20 and 30 Gy doses. However, as 30 Gy was found to be the most effective irradiation dose, it was chosen for studying the radio-protective properties of different compounds. These compounds could increase the bacterial cell protection against X-ray irradiation in concentration-dependent manner. They had a role in stimulation of synthesis or regulation of activity of metal-dependent enzymes, required for reversing the X-ray irradiation damage. The study may prove useful for further estimation of the effectiveness of different compounds as radio-protectors on bacteria and other cells, especially mammalian cells under X-ray irradiation.

Effects of low concentrations of a polychlorinated biphenyl, Aroclor 1254 on membrane bound ion dependent ATPases in mice liver.

Aroclor 1254, a polychlorinated biphenyl, is present in the environment in low concentration but references on its toxic effects on liver cell membrane proteins and the mechanism of actions are not abundantly available. Therefore, the present study was undertaken to investigate the low level, sub-acute dose and exposure duration dependent effects of Aroclor 1254 on total, Na+, K+, Ca2+ and Mg2+-ATPases of the mouse liver. The hypotheses tested in the present study were, (a) whether the low, environmentally available dose and the exposure durations of Aroclor 1254 affects the membrane-bound ion dependent ATPases, and (b) if a response was observed, whether it is a direct or indirect effects of the toxicant. Groups of mice were exposed to different doses (0.1 and 1mg kg-1 body weight d-1) and exposure durations (4 d, 8 d and 12 d) of Aroclor 1254. The results indicated significant exposure duration dependent changes in the specific activity of the selected membrane bound ATPases. As the observed changes were mostly enzyme stimulation after toxication through oral administration, the effects of the Aroclor were possibly indirect, through complex chain of reactions.

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

Effect of methacrylonitrile on membrane bound enzymes of rat brain.

Methacrylonitrile (MeAN) is a plastic monomer. Its effect on membrane bound enzymes like Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, NADH dehydrogenase, alkaline phosphatase (ALP) and various elements like sodium (Na+), potassium (K+), and calcium (Ca2+) in rat brain were studied. Administration of 50 mg/kg body weight/day (0.25 LD50) and 100 mg/kg body weight/day (0.5 LD50) by gavage to rats for 7 days resulted in a significant decrease in activities of Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, and NADH dehydrogenase. A significant reduction in calcium content, potassium content and a significant increase in sodium content and alkaline phosphatase activity in MeAN treated animals were observed. Inhibition of membrane bound enzymes occurred due to either direct effect of MeAN or indirect effect of changes in ionic homeostasis in MeAN treated animals.

Metabolic response of roots to osmotic stress in sensitive and tolerant cereals--qualitative in vivo [31P] nuclear magnetic resonance study.

High resolution [31P] nuclear magnetic resonance (NMR) spectroscopy was used to investigate the changes in phosphate metabolism and intracellular pH in intact root segments of relatively osmotic stress sensitive species maize (Zea mays L) and insensitive species pearl millet (Pennisetron americanum (L) Leeke) exposed to hyper osmotic shock. The results were used to understand the adaptive mechanism of the two species. The hyper osmotic shock resulted in large build-up of phosphocholine and decrease in glucose 6-phosphate (G-6P) and UDPG levels in both the crops. The osmotic shock produced a large vacuolar alkalinization and decrease in pH across tonoplast membrane in maize roots. However, the roots of pearl millet were able to adapt to the stress and maintained pH gradient across tonoplast with marginal vacuolar alkalinization. This may be attributed to the sustained activity of primary tonoplast pumps and increased activity of H+-ATPase that normally maintain pH gradient across tonoplast.

Blue light-sensitive plasma membrane bound exogenous NADH oxidase in Cuscuta reflexa.

Protoplasts isolated from Cuscuta reflexa exhibited a higher rate of exogenous NADH oxidation as compared to NADPH in the dark. NAD(P)H oxidation was monitored by measuring the rate of oxygen consumption and this oxidase system was sensitive to blue light. Both NADH oxidase and its blue light sensitivity were inhibited by -SH group reacting agents. The corresponding changes occurring in H+-extrusion activity and intracellular ATP levels were also monitored. Stimulation of NADH oxidation under blue light corresponded to increased rate of H+-extrusion and intracellular ATP level, the converse was also true under NADH oxidase inhibitory conditions. These observations suggested a close functional association between blue light-sensitive plasma membrane bound redox activity and H+-ATPase in this tissue. Further, concanavalin A binding of protoplasts resulted in a loss in NADH oxidase activity and its blue light sensitivity suggesting apoplastic location and glycoprotein nature of the blue light sensitive NADH oxidase system in Cuscuta.

Effects of decreased cerebral perfusion pressure on cerebral hemodynamics, brain cell membrane function and energy metabolism during the early phase of experimental Escherichia coli meningitis in the newborn piglet

In this study, we tested the hypothesis that decreased cerebral perfusion pressure (CPP) induces cerebral ischemia and worsen brain damage in neonatal bacterial meningitis. Meningitis was induced by intracisternal injection of 10(9) colony forming units of Escherichia coli in 21 newborn piglets. Although CPP decreased significantly at 8 hr after bacterial inoculation, deduced hemoglobin (HbD), measured as an index of changes in cerebral blood flow by near infrared spectroscopy, did not decrease significantly. In correlation analyses, CPP showed significant positive correlation with brain ATP and inverse correlation with brain lactate levels. CPP also correlated positively with HbD and oxidized cytochrome aa3 (Cyt aa3) by near infrared spectroscopy. However, CPP did not show significant correlation with cerebral cortical cell membrane Na+,K+-ATPase activity, nor with levels of lipid peroxidation products. In summary, decreased CPP observed in this study failed to induce cerebral ischemia and further brain injury, indicating that cerebrovascular autoregulation is intact during the early phase of experimental neonatal bacterial meningitis.

Effects of dietary polyunsaturated fatty acids on adenylate cyclase, 5'nucleotidasa and Na+K+-ATPase activities in rat brain-plasma membrane

The incidence of polyunsaturated fatty acids (PUFA) in human nutrition is now generally accepted. As essential membrane components, PUFA may act as enzyme activity modulators. In this study, four different diets, in which PUFA type was the only modifying factor, were evaluated on 5'nucleotidase, adenylate cyclase and Na+/K+ATPase activities in rat brain plasma membranes. Animals fed the total PUFA deficient diet exhibited significant lower body weight and lower brain weight than did the control group. The specific activities of 5'nucleotidase and Na+/K+ATPase in brain plasma membrane were slightly modified by dietary PUFA. The catalytic unit of adenylate cyclase in total PUFA deficient animals presented augmented enzyme activity and animals receiving diets deficient in n-6 PUFA showed reduced activity in relation to the control animals. Our results showed that the epinephrine receptors, in the case of adenylate cyclase are not modified by dietary PUFA, but rather the catalytic unit seems to be altered by dietary PUFA. These results can be partially explained by the fluidity that PUFA confers to membranes facilitating the proximity of enzyme-substrate. The physiological consequences of dietary PUFA incidence on enzyme activity needs further study.

Alteration in the activities of the membrane-integrated enzymes of polymorphonuclear leukocytes in obesity.

Oubain sensitive and insensitive adenosine triphosphatase showed decrease in their activities in the polymorphonuclear leukocytes of obese patients while the activity of acetylcholinesterase was found to be increased significantly. The contents of sodium, potassium and magnesium were found to be significantly decreased in polymorphonuclear leukocytes of obese patients. Polymorphonuclear leukocytes obtained from treated obese patients showed considerable restoration.

Studies on the effect of ethanol on the Na+, and the Na+,K+ATPase activities of plasma membranes of rat kidney proximal tubular cells

In the present work it was investigated the effect of 2 percent ethanol on the Na+ and on the Na+, K(+)-ATPase activities. The differential effect of the alcohol on the two ATPases (approximately 40 percent inhibition of the Na(+)-ATPase and approximately 10 percent inhibition of the Na+, K(+)-ATPase), is not due to a higher degree of denaturalization of the enzyme, nor to a faster effect of ethanol on the Na(+)-than on the Na+, K(+)-ATPase. Our results show that ethanol affects the selectivity of the Na+, K(+)-ATPase for Na+ and/or for K+, enhancing the Na+ affinity for the K+ sites, and/or reducing the K+ affinity for its own sites. This effect was not seen for the Na(+)-ATPase, indicating that 2 percent ethanol inhibits the two ATPases in a totally different way

El aumento del calcio ionizado intracelular depende de algún factor presente en el suero de las pacientes preeclámpticas / The intracellular increase of ionized calcium depend on some factor present in pre-eclamptic patient sera

En trabajos previos nuestro grupo ha demostrado que la concentración intracelular de calcio ionizado es mayor en pacientes con preeclampsia que en embarazadas normotensas y que estas cifras correlacionan con la tensión arterial. Ambos indicadores se normalizan 6 semanas después del parto. Con el fin de investigar la posibile participación de factores solubles en el plasma, en el aumento del calcio libre intracelular, se estudiaron 8 pacientes preeclámpticas diagnosticadad por los criterios del Colegio Americano de Ginecólogos y Obstetras. Como grupo testigo se incluyeron 8 embarazadas normotensas pareadas por edad cronológica y gestacional. Para medir flujos de calcio transmembrana se utilizaron plaquetas de varones sanos. Las plaquetas fueron incubadas durante 0, 15, 30 y 60 minutos en suero de pacientes preeclámpticas o de embarazadas normotensas marcado con Ca. Las diferencias en el transporte de calcio se evaluaron con análisis de varianza de Kruskal Wallis. El transporte de calcio fue mayor cuando las plaquetas se incubaron en suero de pacientes preeclámpticas, Md = 1.475 ñ 0.311 nanomolas que cuando fueron incubadas en suero de embarazadas normotensas, Md = 0.9725 ñ 0.58 nanomolas, p < 0.02. Este hallazgo sugiere que en el suero de las pacientes preeclámpticas existe algún factor que facilicita la entrada de calcio a la célula, que provoca un aumento en la concentración de calcio libre y participa en la hipertensión gestacional.

Interactions of the regulatory ligands Mg2+ and MgATP2- with the renal plasma membrane Ca(2+)-ATPase: effects of osmolytes that stabilize or destabilize protein structure

In this report we analyze the kinetics of activation of the plasma membrane Ca(2+)-ATPase from kidney proximal tubules by the regulatory ligands Mg2+ and MgATP2-, and we examine modifications in the effects of these ligands that are promoted by organic solutes of natural occurrence that stabilize or destabilize protein structure and function. The solutes tested were trimethylamine-N-oxide (TMA-O), sucrose and urea. TMA-O and sucrose were chosen as representative of the different methylamines and polyols, respectively, that accumulate in living organisms. The results lead to the conclusion that free Mg2+ and the MgATP2- complex both activate the rate-determining E2-->E1 transition during the catalytic cycle of the enzyme, by binding to nonidentical and independent regulatory sites. They also indicate that TMA-O, sucrose and urea not only promote global modifications in the enzyme structure, but also modify specific interactions of the ligands Mg2+ and MgATP2- at their regulatory sites

Intracellular calcium homeostasis in Leishmania mexicana. Identification and characterization of a plasma membrane calmodulin-dependent Ca(2+)-ATPase

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...

Role of serine esterase in hydrogen peroxide-mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to hydrogen peroxide cause dose-dependent stimulation of [14C] arachidonic acid (AA) release and enhancement of the cell membrane-associated phospholipase A2 activity as well as of the cell membrane-bound serine esterase activity tested against synthetic substrate p-tosyl-L-arginine methyl ester. While pretreatment of cells with serine protease inhibitors, viz. phenyl methyl sulphonyl fluoride, diisopropyl fluorophosphate and alpha-1-proteinase inhibitor, and antioxidant vitamin E prevents H2O2 stimulation of AA release and the cell membrane-bound serine esterase and PLA2 activities, that with actinomycin D and cycloheximide is devoid of any effect on H2O2 caused stimulation of AA release and the smooth muscle cell membrane associated serine esterase and PLA2 activities. Treatment of the smooth muscle cell membrane suspension with the serine protease trypsin markedly stimulates PLA2 activity. These results suggest that on exposure to H2O2 the smooth muscle cell membrane-bound serine esterase plays an important role in stimulating the cell membrane associated PLA2 activity thereby resulting in an increase in AA release.

Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains.

Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.

Molecular cloning of a membrane bound tyrosine-specific protein kinase from rat spleen.

Tyrosine-specific protein phosphorylation is believed to play an important (though poorly understood) role in various cellular functions in many normal and malignant cells. In order to understand the function of tyrosine-specific protein kinases in normal cells, it is necessary, as an initial step, to identify genes (and proteins) for these enzymes. For this purpose cDNA libraries were constructed in plasmid vector pGEM-3Z and lambda gt11 using mRNA from rat spleen. From these cDNA libraries, cDNA clones coding for a src-related tyrosine-specific protein kinase were isolated. The largest clone (L115) was 1.94 kb in size. Various restriction fragments of this clone were subcloned in plasmid vector for sequencing. The complete nucleotide sequence of the largest clone showed an open reading frame coding for a protein of 503 amino acids. The presence of a glycine at position 2 and an arginine at position 7 indicated that this protein is likely to be acylated at glycine 2 and therefore associated with plasma membrane. This gene showed high homology to human and mouse hck and hence it is perhaps the rat homologue of hck. Moderate level of expression of this gene was observed only in the adult rat spleen and not in other tissues. These results suggest that this kinase gene is expressed in a tissue specific manner.