Effects of amino acids and derivatives of cyclic adenosine 3′, 5′-monophosphate on the production of three exoenzymes by Staphylococcus, simulans biovar staphylolyticus

The extracellular protease, endopeptidase, and hexosaminidase produced by $\text{Staphylococcus}\text{,}\text{simulans}\text{biovar}\text{staphylolyticus}$ were neither induced nor repressed by amino acids but required a tryptic digest of casein for their production. Catabolite repression of exoenzyme production by glucose was not affected by exogenous cyclic adenosine 3′, 5′-monophosphate but was partially relieved by di- or monobutyryl derivatives of this compound.     

The Unique Chemistry of Eastern Mediterranean Water Masses Selects for Distinct Microbial Communities by Depth

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of UDP-Glucose:(1,3)-beta-Glucan Synthase to Inactivation by Phospholipases and Trypsin

UDP-glucose:(1,3)-β-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A₂. Enzyme activity could not be restored to the phospholipase-treated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques

The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes.     

Ion exchange process for removing glucosyltransferase from crude glucoamylase

The advant of a new range of high-protein capacity cellulosic ion exchangers suitable for use on an industrial scale made it worthwhile investigating the conditions necessary to remove the contaminating enzyme glucosyltransferase from a commercial preparation of crude glucoamylase. The potential of the SP derivative, SP Indion((R)), for achieving this separation is shown. At pH 2.5 the glucosyltransferase was selectively adsorbed by the ion exchanger, and 99% of the glucoamylase was recovered in the eluate from the column. Purification of an Aspergillus culture filtrate by this method will require careful control of the ionic strength of the culture medium if it is to be used without the additional step of cation exchange to lower than pH to 2.5.     

Exudation of amino acids by intact and damaged roots of wheat and peas

Summary Wheat and pea seedlings were grown aseptically in solution-culture and the total free amino nitrogen released by the roots was determined by a quantitative ninhydrin test. Amino nitrogen from wheat plants after 14 days growth was not detected by the test, indicating the release of less than 3 µg of amino nitrogen from a culture of 15 plants. Pea plants of the same age released from 2 to 7 µg per plant. Paper chromatograms of highly concentrated undisturbed solution-cultures revealed up to 13 amino compounds from wheat and 11 from pea. The pattern of amino acids in exudates was similar to that in crushed roots, except for an unidentified amino compound which was detected only in exuded material. The total amino nitrogen and relative proportions of several amino acids in the root exudates of sand-grown peas was influenced by several ratios of oxygen and carbon dioxide supplied to the root zone. Roots, experimentally damaged by swirling and rinsing in sand, released in 1 hour amino nitrogen of from 73 to 120 per cent of that released by normal exudation over a 2-week period. Our findings suggest that experimental and environmental root damage may be responsible for a large proportion of organic materials released by growing plant roots.Trade names are used in this publication to provide specific information. Their use does not constitute a guarantee of the products named and does not signify that they are approved by the U.S. Department of Agriculture to the exclusion of others of suitable composition.     

Accuracy of pulse oximetry to estimate HbO2 fraction of total Hb during exercise

The accuracy of two pulse oximeters (Ohmeda 3700 and Biox IIa) was evaluated during cycle ergometer incremental exercise in 10 healthy subjects. The exercise protocol began at 30 W with the power output being increased 15 W.min-1 until volitional fatigue. Ear and finger probe pulse oximetry measurements of available hemoglobin (%Spo2) were compared with arterial oxyhemoglobin fraction of total hemoglobin (%HbO2) measured directly from arterial blood samples using a CO-oximeter. To provide a wide range of %HbO2 values, four subjects exercised under hypoxic conditions [inspired partial pressure of O2 (PIo2) = 107 Torr], while the remaining six subjects exercised under normoxic conditions (PIo2 = 150 Torr). Because carboxyhemoglobin (HbCO) or methemoglobin (MetHb) is not measured by pulse oximeters, %HbO2 was corrected for HbCO and MetHb and expressed as percent arterial O2 saturation of available Hb (%Sao2). Small and insignificant differences (P greater than 0.05) existed between SpO2 (all 3 instruments) and %SaO2 at the lowest work rate and the highest power output achieved. Regression analyses of %SpO2 vs. %SaO2 produced correlation coefficients of r = 0.82 [standard error of the estimate [(SEE) = 1.79], r = 0.89 (SEE = 1.48), and r = 0.93 (SEE = 1.14) for the Biox IIa, Ohmeda 3700 (ear), and the Ohmeda 3700 (finger) pulse oximeters, respectively. We conclude that pulse oximetry, within the above limits of accuracy, is useful in estimating %SaO2 during exercise in healthy subjects.