Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Copper metabolism in the plaice, Pleuronectes platessa (L.)

Copper metabolism in a teleost, the plaice, Pleuronectes platessa (L.) from a natural environment has been studied. Distribution in the various tissues of the metal and of five key copper-dependent enzymes: ceruloplasmin EC 1.16.3.1; Superoxide dismutase EC 1.15.11; tryptophan oxygenase EC 1.13.1.12; cytochrome oxidase EC 1.9.3.1 and monoamine oxidase EC 1.4.3.4 have been determined. The copper distribution was found to be similar to that in mammals with the greatest concentrations in the brain and heart. Distribution of the copper enzymes is also similar to that found in mammals. A preliminary characterization of the copper enzymes showed that plaice cytochrome oxidase has a pH maximum 2 pH units more alkaline than the mammalian enzyme and that plaice tryptophan oxygenase is more sensitive to heat denaturation than the mammalian enzyme. The present data form a base-line against which studies on factors affecting the copper metabolism in a teleost can be assessed.     

Trace analysis of the MIF analogue pareptide in blood plasma by high-performance liquid chromatography and short-wavelength excitation fluorometry

A high-performance liquid chromatographic procedure was developed and applied to analysis of the pharmacologically active MIF analogue pareptide in human plasma. The procedure involves formation of a fluorescent 7-chloro-4-nitrobenzyl-2-oxa-1,3-diazole-(NBD-CI) pareptide derivative followed by separation of the NBD derivative from plasma components on a 30-cm microparticle octadecylsilane bonded column. The separated derivative was quantitated using a short-wavelength excitation fluorometric detector. The detection limit of pareptide in plasma samples was 5 ng or 17 pmoles per ml of plasma. In the absence of plasma, the corresponding on-column detection limit was 0.5 pmoles.     

Viral Pollution of Surface Waters Due to Chlorinated Primary Effluents

The role of chlorinated primary effluents in viral pollution of the Ottawa River (Ontario) was assessed by examining 282 field samples of wastewaters from two different sewage treatment plants over a 2-year period. The talc-Celite technique was used for sample concentration, and BS-C-1 cells were employed for virus detection. Viruses were detected in 80% (75/94) of raw sewage, 72% (68/94) of primary effluent, and 56% (53/94) of chlorinated effluent samples. Both raw sewage and primary effluent samples contained about 100 viral infective units (VIU) per 100 ml. Chlorination produced a 10- to 50-fold reduction in VIU and gave nearly 2.7 VIU/100 ml of chlorinated primary effluent. With a combined daily chlorinated primary effluent output of approximately 3.7 × 10⁸ liters, these two plants were discharging 1.0 × 10¹⁰ VIU per day. Because the river has a mean annual flow of 8.0 × 10¹⁰ liters per day, these two sources alone produced a virus loading of 1.0 VIU/8 liters of the river water. This river also receives at least 9.0 × 10⁷ liters of raw sewage per day and undetermined but substantial amounts of storm waters and agricultural wastes. It is used for recreation and acts as a source of potable water for some 6.0 × 10⁵ people. In view of the potential of water for disease transmission, discharge of such wastes into the water environment needs to be minimized.