Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Polymerization studies on protein from the PM2 strain of tobacco mosaic virus: Light scattering and related studies

Light-scattering and related studies on the polymerization behavior of the protein from the PM2 strain of TMV show that in phosphate buffer of ionic strength 0.1, the maximum extent of temperature-mediated polymerization occurs at pH values lower than in the case of TMV protein. The pH range of temperature-induced polymerization is from 5.0 to 6.0, contrasted with 5.0 to 7.5 for TMV protein. Velocity sedimentation studies show that PM2 protein at room temperature in phosphate buffer (I = 0.1) has sedimentation coefficients of 174 S, 104 S, and 4.3 S at pH values of 4.89, 5.53, and 7.5. Electron microscope studies show that at room temperature in phosphate buffer of 0.1 ionic strength at pH 5.53, PM2 protein has structures resembling essentially that of stacked double discs with an occasional helical structure. Similar studies of PM2 protein in 0.1 M ammonium acetate buffer at pH 5.2 show single, double, and double-double helices.     

Effect of osmotic potential, pH, and temperature on the growth of a helical, motile mycoplasma causing corn stunt disease.

Growth characteristics of corn stunt spiroplasma, a helical, motile mycoplasma, were studied over a range of osmolality, pH, and temperature in a simple medium containing 20% (v/v) agamma horse serum, 1.5% (w/v) PPLO broth, and various concentrations of sucrose. The spiroplasma was able to grow in a wide spectrum of osmolalities from 360 to 1120 mosm. Optimal growth was observed in media that contained 0.25-0.35 M sucrose. The organism became longer and thinner in media adjusted to 0.65 M sucrose or more. The spiroplasma lost helicity and motility immediately after transfer to media at pH 5.4 or lower. Optimal pH for growth was 7.2. No growth was observed at pH lower than 5.4 or higher than 8.0. Optimal temperature for growth was 32 degrees C. Very little or no growth was observed at temperatures lower than 15 degrees C or higher than 35 degrees C.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes.

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Growth and division of spiroplasmas: morphology of Spiroplasma citri during growth in liquid medium.

The helical mycoplasma Spiroplasma citri was examined by electron microscopy with a newly developed transfer technique which preserves the helical morphology of the organism. The smallest viable cell was found to be a two-turn (elementary) helix. During the logarithmic phase of growth, organisms increased in length and divided by constriction, liberating two-turn elementary helices. The most frequently dividing parental helix was one with approximately four turns, yielding two elementary helices. Influence of pH and temperature on the morphology of the organism was also investigated. In unbuffered medium, growth of the organism produced a significant decrease in pH and a consequent formation of abnormal morphological forms and cell lysis. At 37 degrees C, cell division was inhibited, leading to a progressive disappearance of two-turn helices and an increase in the average length of other helices. Finally, helices were never seen to arise from round bodies at any stage of the growth cycle.     

Transfer of cobalamin from intrinsic factor to transcobalamin II

The process is obscure by which cobalamin (Cbl) in the endocytosed intrinsic factor (IF)-cobalamin (Cbl) complex is released and transferred to transcobalamin II (TCII) within the enterocyte. Using recombinant IF and TCII, binding of Cbl to IF at pH 5.0 was 70% of binding at pH 7.0, whereas for TCII alone, the value was only 12%. TCII binding activity was lost rapidly at lower pH, but this was not due to protease action. TCII incubated at pH 5.0 with cathepsin L was degraded and could not subsequently bind Cbl. Thus, transfer from IF to TCII is unlikely to occur within an acid compartment. Only 13-15% of bound Cbl was released at pH 5.0 and pH 6.0 from either rat IF, human IF, or human TCII. The K(a) of human or rat IF at pH 7.5 was 2.2 nM; for TCII, the value was 0.34 nM. At pH 7.5, Cbl transfers from IF to TCII, but only to a limited extent (21%), as detected by nondenaturing electrophoresis. Transfer of Cbl from IF to TCII could not be demonstrated at pH values of 5.0 or 6.0. Thus, luminal transfer of Cbl between IF and TCII is likely to be limited, but is possible. The most likely mechanism for intracellular transfer of Cbl from IF to TCII involves initial lysosomal proteolysis of IF, with subsequent Cbl binding to TCII in a more neutral cellular compartment.     

Growth and division of Spiroplasma citri: elongation of elementary helices.

The smallest viable cell of Spiroplasma citri is a two-turn helix (elementary helix). This elementary helix grows into longer parental cells, which then divide by constriction. The helical morphology is conserved during this process. The growth pattern of S. citri membranes has been investigated by different methods of membrane labeling. When labeling is done with specific antibodies, a diffuse growth of the membrane is observed. On the contrary, pulse-labeling of the membrane with tritiated amino acids reveals a polar growth of the organism. Finally, labeling of oxydo reduction sites with potassium tellurite also indicates a polarity in the organism. These results are discussed, and a scheme for spiroplasma growth is proposed.     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Preparation and characterization of microencapsulated proteinase inhibitor aprotinin.

Preparation of microcapsules through interfacial cross-linking of soluble starch/hydroxyethyl starch and bovine serum albumin (BSA) with terephthaloyl chloride is described. The proteinase inhibitor aprotinin, either native or active site protected, was microencapsulated, being incorporated in the aqueous phase. The influence of aqueous phase pH, BSA, and terephthaloyl chloride concentrations as well as stirring rate on microcapsule morphology and size was studied. The polycondensation pH was shown to be the determining factor for tough microcapsule production with a high encapsulation yield. The size of the microcapsules ranged between 10-30 and 50-100 microm at stirring speed 1500 and 500 rpm, respectively. Fourier transform infrared spectroscopic studies were performed on microcapsules prepared under various conditions. A correlation was established between spectral changes and microcapsule morphology and size. The optimal conditions for microcapsule degradation by alpha-amylase were found. Active site-protected aprotinin was shown to fully retain its activity after microencapsulation.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.     

Effect of carbohydrates and control of culture pH on beta toxin production by Clostridium perfringens type C.

Clostridium perfringens type C strain CN 5384 produced a higher level of beta toxin in a controlled pH medium containing 1% glucose, starch, or sucrose than in media with dextrin, fructose, or raffinose. Toxin synthesis was not related to the growth yield. The effect of glucose on beta toxin production by 11 strains was investigated with and without control of the culture pH at 7.5. Strain CN 5386 produced distinctly higher toxin when the pH of the culture was maintained at 7.5, compared with uncontrolled pH.     

Effects of pH on biomass, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture

Three cutaneous propionibacteria, Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum, were grown in chemostats using semi-synthetic medium at various pH values. Growth occurred between pH 4.5 and 7.5 for P. acnes and pH 5.0 and 8.0 for P. avidum and P. granulosum. The highest mumax was at pH 6.0 for the three species. Maximum biomass production was obtained at pH 6.0 for P. acnes and P. avidum and at pH 7.5 for P. granulosum. Extracellular enzyme production occurred over the entire pH growth range when denaturation of the enzymes was taken into account. However, detectable activity of the enzymes was found in a narrower range of pH due to the denaturation of the enzymes at low or high pH values. The highest production of enzymes occurred at pH values between 5.0 and 6.0, apart from the production of hyaluronate lyase of P. granulosum (pH 6.0 to 7.0) and the proteinase of P. acnes and P. avidum (pH 5.0 to 7.5). Propionibacterium acnes produced a lipase, hyaluronate lyase, phosphatase and proteinase activity. Propionibacterium avidum produced a lipase and proteinase activity. Propionibacterium granulosum produced a lipase and hyaluronate lyase.     

Interactions of urocanic acid with bovine serum albumin and the influence of pH on binding affinities: a docking simulation study

In the present study, the interactions of urocanic acid (UA) with bovine serum albumins (BSA) at pH 5.0 and 7.4 were investigated by means of docking simulations. The binding modes of trans- and cis-UA to BSA at pH 5.0 and 7.4 were analysed. In addition, the theoretically predicted binding abilities of zwitterion and anion of UA with BSA are in good agreement with the experimental results. Through comparison with the binding patterns, we revealed that the stronger interactions of UA anion with BSA relative to the zwitterion primarily result from: (1) the increased number of hydrogen bonds between UA anion and BSA; (2) the attractive electrostatic interaction between the deprotoned carboxyl group in UA anion and Arg433 in comparison with the repulsion between the imidazole moiety in zwitterion and the same residue in BSA. This provides a rational explanation for the experimental finding that the binding of UA to BSA at pH 7.4 is much stronger than at pH 5.0.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.