Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

On the mechanism of action of lysophospholipase-transacylase from rat lung

Lysophospholipase-transacylase from rat lung catalyzes the transfer of palmitate from 1-palmitoyl-$\text{sn}$-glycero-3-phosphocholine to water and to another molecule of 1-palmitoyl-$\text{sn}$-glycero-3-phosphocholine. Incorporation of palmitate into phosphatidylcholine is restricted to palmitate donated by lysophosphatidylcholine, free palmitate cannot be esterified to lysophosphatidylcholine by the enzyme. Experiments in the presence of H₂¹⁸O and mass spectrometric analysis of the reaction products show that ¹⁸O is incorporated into the released palmitate but not into the transesterification product phosphatidylcholine. This proofs that the hydrolytic reaction proceeds by O-acyl cleavage. Furthermore, the results strongly suggest that transfer of palmitate to lysophosphatidylcholine occurs through an intermediary covalent acyl-enzyme complex.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

Chemical modification of opiate receptors with ethoxyformic anhydride and photo-oxidation: evidence for essential histidyl residues

In the presence of $\text{D}\text{=}$-carnitine significant decarboxylation of 2-oxoglutarate occurs with γ-butyrobetaine hydroxylase (EC 1.14.11.1) both from $\text{Pseudomonas}$ sp AK 1 and from human kidney. No product was formed from carnitine when $\text{D}\text{=}\text{L}\text{=}$-carnitine was incubated with either enzyme but succinate was formed in 1:1 stoichiometry to decarboxylation using $\text{D}\text{=}$-carnitine and the human enzyme. $\text{L}\text{=}$-Carnitine is also an uncoupler for the human enzyme. There is no significant decarboxylation of 2-oxoglutarate in the absence of a substrate, but during normal catalysis in the presence of γ-butyrobetaine the formation of CO₂ from 2-oxoglutarate exceeds carnitine formation with 20% for the human enzyme.     

Regulation of muscle phosphorylase activity by carnosine and anserine

Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase $\text{a}$ in the presence and absence of AMP and phosphorylase $\text{b}$ in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase $\text{b}$ showing a greater degree of activation than phosphorylase $\text{a}$. Anserine (β-alanyl-L-N^π-methyl-histidine) activates phosphorylase $\text{a}$ to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase $\text{b}$ a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase $\text{a}$ in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase $\text{b}$ by anserine is also observed in the presence of these inhibitors.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

A Novel function of cytochrome C (555, Chlorobium thiosulfatophilum) in oxidation of thiosulfate

Thiosulfate-cytochrome c-551 reductase derived from $\text{Chlorobium}\text{thiosulfatophilum}$ has been highly purified. The enzyme reduces cytochrome $\text{c}\text{-551 of}\text{C}\text{.}\text{thiosulfatophilum}$ in the presence of thiosulfate while cytochrome $\text{c}$-555 of the organism is not reduced by the enzyme. Cytochrome $\text{c}$-555 reacts with the enzyme at an appreciable rate only in the presence of cytochrome $\text{c}$-551. However, the reduction rate of cytochrome $\text{c}$-551 by the enzyme is greatly enhanced on addition of a catalytic amount of cytochrome $\text{c}$-555. Therefore, cytochrome $\text{c}$-555 seems to function as an effector on thiosulfate-cytochrome $\text{c}$-551 reductase as well as it acts as the electron donor to the light-excited chlorobium chlorophylls.     

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Arginyl-tRNA-protein transferase in eucaryotic protists

Soluble extracts of $\text{Saccharomyces cerevisiae}$ and $\text{Blastocladiella emersonii}$ were found to catalyze the specific transfer of arginine from a mixture of [¹⁴C] aminoacyl-tRNAs into protein. Arginine transfer was stimulated by bovine serum albumin. Glu-Ala, Asp-Ala and cystinyl-$\text{bis}$-Ala inhibited incorporation into protein, whereas dipeptides with other NH₂-terminal residues linked to alanine did not. These results indicate the presence of an enzyme in eucaryotic protists with the same donor and acceptor specificity as mammalian arginyl-tRNA-protein transferase.     

S-adenosylhomocysteine is bound to pineal hydroxyindole o-methyltransferase

A substance with maximal absorbance at 260 nm was co-chromatographed with hydroxyindole O-methyltransferase (EC 2.1.1.4) by immunoaffinity chromatography. The co-chromatographed substance was separated from the transmethylase by stepwise elution and was identified as S-adenosylhomocysteine by spectrophotometrical analysis, and by thin-layer chromatography. Identity of S-adenosylhomocysteine was confirmed by determination of demethylated product by using a mixture of [carboxyl-¹⁴C]S-adenosylmethionine and [methyl-³H]S-adenosylmethionine as a substrate. The immunoaffinity chromatography provides direct evidence for a presence of the enzyme-product complex $\text{in vivo}$ and $\text{in vitro}$. At low concentration of S-adenosylmethionine enzymatic activity was inhibited by the co-purified S-adenosylhomocysteine. The endogenous S-adenosylhomocysteine bound to the enzyme probably regulates the melatonin biosynthesis $\text{in vivo}$.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.