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PubMed1984

Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.).

The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal.

Animals↗
PubMed1996

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

Crystallization↗
PubMedDate not supplied

Novel approaches to the purification of penicillin acylase.

Penicillin acylase of E. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0-7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purified enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.

Amidohydrolases↗
PubMed1975

[15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization].

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.

Alcohol Oxidoreductases↗
PubMed1979

Electrical conductance of directly compressible materials under pressure.

An electrometer that allowed direct recording of resistance changes during compression was used to measure the conductances of sodium chloride, potassium chloride, ammonium chloride, and sodium citrate. The effects of initial particle size, lubricant level, moisture content, and compaction pressure were determined. Direct compression tableting diluents also studied were dextrose, sucrose, lactose, microcrystalline cellulose, and dibasic calcium phosphate. Distinctive conductance patterns were observed and are rationalized in terms of previously proposed bondign machanisms.

Electric Conductivity↗
PubMed1999

Comparative effects of ammonium and nitrate compounds on Pacific treefrog and African clawed frog embryos.

The effects of ammonium nitrate, ammonium chloride, ammonium sulfate, and sodium nitrate on survival and growth of Pacific treefrog (Pseudacris regilla) and African clawed frog (Xenopus laevis) embryos were determined in static-renewal tests. The 10-day LC50s for the three ammonium compounds for P. regilla ranged from 25.0-32. 4 mg/L NH4 -N. The 10-day sodium nitrate LC50 for P. regilla was 578. 0 mg/L NO3-N. LC50s for X. laevis exposed for 4 or 5 days to the three ammonium compounds ranged from 27.5-60.2 mg/L NH4-N. The sodium nitrate LC50 for X. laevis ranged from 438.4-871.6 mg/L NO3-N. The lowest LOAEL based on length or weight was 6.1 mg/L NH4-N for the two species. The lowest LOAELs for NO3-N were 111.1 mg/L for P. regilla and 56.7 mg/L for X. laevis. Calculated unionized NH3 comprised 0.5-1.8% of measured NH4-N concentrations. Potential harm to amphibian populations could occur if NH4-N and NO3-N in agricultural runoff or drainage impacts sensitive life stages for a sufficiently long period.

Animals↗
PubMed2004

Beta-carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea.

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3' R-beta, beta-carotene-3, 3' diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of beta-carotene (beta, beta-carotene), beta-cryptoxanthin (beta, beta-caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on beta-carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and beta-carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l(-1) urea and sodium carbonate, respectively. The maximum beta-carotene level was 7.85 microg ml(-1) culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific beta-carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that beta-carotene was produced maximally during the late exponential phase at 0.41 microg ml(-1) h(-1). It is possible that this organism could be an excellent commercial source of either beta-carotene or zeaxanthin, depending on initial culture conditions.

Culture Media↗
PubMed2001

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources.

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.

Bacillus megaterium↗
PubMed1996

Responses of Sarcoptes scabiei (Acari: Sarcoptidae) to nitrogenous waste and phenolic compounds.

This study found that > or = 1 life stage(s) of Sarcoptes scabiei (L.) was significantly attracted to the nitrogenous compounds, guanine, purine, adenine, allantoin, hypoxanthine, xanthine, uric acid, ammonium chloride, ammonium nitrate, and ammonium sulfate. Females and larvae responded to all compounds. Males responded to all compounds except purine and allantoin. Nymphs responded to all compounds except allantoin. Some specific concentrations of compounds attracted only 1 life stage but other concentrations attracted multiple life stages. Also, some life stages were attracted by multiple concentrations of a compound. The concentration that induced the greatest response by a particular life stage varied between life stages. Overall, greater percentages of females were attracted to the nitrogenous compounds than any other life stage. Females responded to more concentrations of a compound compared to other life stages. Males responded to fewer concentrations then immatures. In addition, all life stages of S. scabiei were significantly attracted to the phenolic compounds, 2,6-dichlorophenol, methyl salicylate, and 2-nitrophenol. Nymphs followed by females responded to the most concentrations. The results indicated that these nitrogenous wastes and phenolic compounds acted in a pheromone-like manner that induced assembly of these ectoparasitic mites.

Animals↗
PubMed1969

Control of dimorphism in a biochemical variant of Candida albicans.

The cellular morphology of a biochemical variant of Candida albicans could be controlled by the ratio of carbon dioxide to oxygen in the culture system or by individual amino acids. Predominantly pseudohyphal morphology was observed (i) at a CO(2) to O(2) ratio of 2:1 and (ii) without the addition of carbon dioxide, when either glycine, d- or l-ornithine, l-serine, l-methionine, l-phenylalanine, or l-tyrosine was the sole nitrogen source in the culture medium. When ammonium chloride, ammonium sulfate, l-glutamic acid, l-glutamine, or l-proline was the nitrogen source, yeastlike growth was observed in the presence or absence of CO(2). More adenosylmethionine was present in pseudohyphal than in yeastlike cells, and pseudohyphal cell wall preparations contained less methionine than cell walls from the yeastlike form. These results suggest a correlation between sulfur amino acid metabolism and dimorphism.

Amino Acids↗
PubMed1992

Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae.

The effect on D-xylose utilization and the corresponding xylitol and ethanol production by Candida shehatae (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(p/s), yield coefficient (grams product/grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethanol:xylitol ratio (11.0) among all the nitrogen sources tested. The ratio of NADH- to NADPH-linked D-xylose reductase (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose.

Aldehyde Reductase↗
PubMed1996

Acceptance of minerals and other compounds by calcium-deprived rats: 24-h tests.

We measured 24-h spontaneous intake of four to eight concentrations of 31 different solutions by groups of rats fed control or low-calcium diets. Relative to controls, those fed low-calcium diet had increased acceptance of one or more concentrations of sodium chloride, sodium acetate, and sodium bicarbonate, but not sodium gluconate. Differences in palatability between these sodium salts were unimportant because the rats fed low-calcium diet consumed more sodium chloride even if this was made less acceptable by adulteration with citric acid. The possibility that calcium-deprived rats have an enhanced general cation or mineral appetite was supported by findings of increased acceptance of one or more concentrations of nine of ten chloride minerals tested (aluminum chloride, ammonium chloride, ferric chloride, ferrous chloride, magnesium chloride, sodium chloride, potassium chloride, strontium chloride, zinc chloride). However, there were no differences in acceptance of any concentration of cesium chloride, magnesium sulfate, or lead acetate. Moreover, calcium-deprived rats drank more hydrochloric acid and malic acid than did controls. Thus the effect of calcium deficiency on intake was not confined to minerals. Acidity or bitterness did not appear important because there was no difference between the groups in intake of sulfuric acid, citric acid, or quinine hydrochloride. Consistent with the exacerbating effects of phosphates on calcium deprivation, deprived rats had decreased intakes of phosphates (sodium phosphate, potassium phosphate). However, they also had decreased intakes of sucrose and saccharin. It is clear that calcium deprivation does not induce a general increase in acceptance of all taste solutions, but there appears to be no simple explanation for what these animals consume.

Acids↗