Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Survival of ram testicular spermatozoa invitro: Effects of glucose,glucose metabolites,rete testis fluid-proteins,selected androgens and phospholipids

The effects of glucose, glucose metabolites, protein-enriched rete testis fluid (RTF), selected androgens and phospholipids on the survival of testicular spermatozoa $\text{in}$$\text{vitro}$ have been studied. The oxidative and glycolytic activity, motility and percentage of live cells were the criteria for assessing the viability of the spermatozoa washed free of the substances that had been added during storage. The addition of lithium lactate, sodium lactate and sodium pyruvate but not glucose was beneficial to the survival of testicular spermatozoa. Following 10 to 12 days storage at 4°C with added lactate or pyruvate testicular spermatozoa had a higher glycolytic activity than did control spermatozoa. The respiratory activity of stored testicular spermatozoa was maintained or depressed, depending on the density of spermatozoal suspension during storage. After 10 to 11 days storage in concentrated RTF or after exposure to selected androgens testicular spermatozoa utilized more glucose than after storage with lactate alone. However, this apparent response to androgens and RTF-proteins was the consequence of a higher survival rate of the spermatozoa rather than an increase in the metabolic activity of individual spermatozoa. These results indicate that metabolites of glucose may serve as substrates for spermatozoa in the epididymis while certain androgens and macromolecules occurring in reproductive tract fluids may play important roles in the survival of spermatozoa during their period of maturation in the epididymis.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Analysis of the inhibitor binding to the nucleoside site of phosphorylase b

The binding of inhibitors to site I of rabbit muscle phsphorylase $\text{b}$ has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both $\text{Δ}\text{H}$ and $\text{Δ}\text{S}$ of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase $\text{b}$ is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive $\text{T}$ conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active $\text{R}$ conformation, whereas non-exclusive binding of the activator was observed when caffeine was added.     

Fatty acid hydroperoxide lyase in tobacco cells cultured in vitro

Fatty acid hydroperoxide lyase (HPO lyase) was found in green and non-green tobacco cells cultured in vitro. The HPO lyase activity in non-green cells was $\text{1}\text{3}$-$\text{1}\text{2}$ of that in green cells. When the cells were transferred from the light to dark conditions or vice versa, cells turned non-green or green according to the light conditions. The HPO lyase activity also changed according to the light conditions, but the changes in HPO lyase activities were not proportional to the changes in chlorophyll contents. These results suggest that at least two types of HPO lyases are present in the green cells. One type of HPO lyase is perhaps common both to the green and non-green cells; another one is chloroplastic. The fatty acid compositions of cells and substrate specificities of HPO lyase differed between green and non-green cells.     

Effect of carbamyl phosphate on the regulation of nitrogenase in Clostridium pasteurianum

One mM carbamyl phosphate inhibited the $\text{in}$$\text{vitro}$ acetylene reduction activity of nitrogenase 30% whereas at high concentrations a maximum inhibition of 50% was observed. When 1 mM carbamyl phosphate was added to a culture growing of N₂ 1) nitrogenase synthesis was completely repressed and 2) after a period of 2.5 hrs in the absence of growth, the specific activity decreased to less than 50% of its activity just before the addition of the inhibitor.     

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A₂ activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from $\text{E.}$$\text{coli}$ membranes. Phospholipase A₂ activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A₂ activity which was assayable in islet homogenates. Glucose stimulated phospholipase A₂ in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A₂ activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A₂ activity measured in the presence of high calcium concentrations. Phospholipase A₂ activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A₂ activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.     

Induction by retinoic acid of NAD+-glycohydrolase activity of myelomonocytic cell lines HL-60, THP-1 and U-937, and fresh human acute promyelocytic leukemia cells in primary culture

HL-60, a human promyelocytic leukemia cell line, is induced to differentiate by retinoic acid to mature granulocytes. We have now found that after the addition of 1 μM retinoic acid to HL-60 cultures an increase in NAD⁺-glycohydrolase (NADase) activity is detected by 6 hr and after a 33-fold increase in activity reaches a plateau by 24 hr. Cycloheximide inhibits completely the retinoic acid-induced increase in NADase activity indicating that enzyme induction requires protein synthesis $\text{de}\text{,}\text{novo}$. An increase of NADase activity was found not only in HL-60 cells but also in two human monoblast cell lines (U-937 and THP-1) and fresh cells in primary culture from two patients with acute promyelocytic leukemia. An increase in synthesis $\text{de}\text{,}\text{novo}$ of NADase does not appear to be obligatory for differentiation of HL-60 because there was no increase of NADase activity in HL-60 cells induced to differentiate with either dimethylsulfoxide, hypoxanthine, butyrate, or 1, 25-dihydroxycholecalciferol and there were marked increases in NADase activity at concentrations of retinoic acid having little or no effect on differentiation.     

Evidence against the presence of cyclic AMP and related enzymes in selected strains of Bacteroides fragilis

Cyclic AMP was not detected in whole cells, expended culture medium or culture supernatant fluid of selected strains of $\text{Bacteroides fragilis}$. Adenyl cyclase and c-AMP phosphodiesterase activities were also not detected in cell extracts of $\text{B. fragilis}$. The exogenous addition of dibutyryl-c-AMP or sodium cholate to cultures of $\text{B. fragilis}$ growing on lactose did not significantly affect the specific activity of β-galactosidase measured in cell extracts of this organism. No diauxic growth pattern could be demonstrated in a chemically defined medium containing 5 mM glucose + 28 mM lactose.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

Evidence for the oxidation of glycolytic NADH by the malate-aspartate shuttle in Ehrlich ascites tumor cells.

Actively glycolyzing Ehrlich-Lettré ascites tumor cells have been tested for their ability to reoxidize into the mitochondria reduced nicotinamide adenine dinucleotide by the malate-aspartate shuttle. The aspartate transaminase inhibitor aminooxyacetate has been used as a tool for evaluating it. Measurements of the free cytosolic $\text{(NADH)}\text{(}\text{NAD}{}^{\mathrm{+}}\text{)}$ ratio indicate that it increases gradually in the inhibitor-treated cells up to a value about ninefold higher than in the controls after 30 min of glycolytic activity. Fructose 1, 6-diphosphate and dihydroxyacetone phosphate reach a steady-state level after 5 min of incubation in the untreated cells, whereas they accumulate in large amounts in the inhibited cells. Correspondingly, a decrease in 3-phosphoglycerate concentration is observed. On the other hand, the rate of glucose utilization is affected slightly only during long observation times. From these results it may be established that in Ehrlich ascites cells reducing equivalents generated in the cytosol during aerobic glycolysis strongly influence the NAD redox state when their intramitochondrial translocation is prevented by the inactivation of the malate-aspartate shuttle.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with $\text{bis-(}\text{p-}\text{nitrophenyl)}$ phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards $\text{5′ -}\text{dTMP}\text{-p-}\text{nitrophenyl}$ ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

The exchange and maximal net flux of glucose across the human erythrocyte. II. The effect of two sulphydryl enzyme inhibitors,chlormerodrin and p-chloromercuribenzene sulfonic acid

The exchange and maximal net fluxes of [¹⁴C]glucose across the membrane of the human red cell were measured. The effects of $\text{p-}\text{chloromercuribenzene}$ sulfonic acid and chlormerodrin on these two parameters of glucose transport were determined. At low concentrations $\text{p-}\text{chloromercuribenzene}$ sulfonic acid (a non-penetrating organic mercurial) was found to readily inhibit the exchange flux but not the net efflux. At extremely high concentrations of $\text{p-}\text{chloromercuribenzne}$ sulfonic acid the maximal net efflux showed some degree of inhibition. Chlormerodrin (a penetrating organic mercurial) inhibited both the exchange and net fluxes in the same manner. The addition of insulin in certain instances reduced the degree of inhibition caused by the organic mercurials. Insulin had no effect on the amount of either $\text{p-}\text{chloromercuribenzene}$ sulfonic acid or chlormerodrin which bound to the red cell. From the results obtained, it is suggested that there exist glucose-reactive sites on both the outer and inner surfaces of the membrane. The results also suggest a carrier system possessing different sites or molecular arrangements for glucose egrees and for glucose entry.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.     

Presence of glycerol and fatty acids in the C-terminal end of a variant surface glycoprotein from Trypanosoma equiperdum

The composition of the C-terminal end of a variant surface glycoprotein from $\text{Trypanosoma equiperdum}$ (BoTat-1 VSG) has been examined. It has been reported for two $\text{Trypanosoma brucei}$ VSGs (Holder, A.A., Biochem. J. (1983), $\text{209}$, 261–262) that ethanolamine was involved in binding the C-terminal amino acid to an oligosaccharide side chain. Tryptic glycopeptides were prepared from BoTat-1 VSG and analyzed. One of them was found to contain ethanolamine and consequently was assumed to be C-terminal. It was shown that the glycopeptide also included phosphate, glycerol and fatty acids. The fatty acid composition was representative of that of glycerolipids. All the results suggest that the end of the molecule is a core of phosphatidylethanolamine.