Effects of Phosphate Residues on the Excellent Emulsifying Properties of Phosphoglycoprotein Phosvitin

The effects of phosphate residues in phosvitin on its emulsifying properties were investigated. The emulsifying properties, especially the emulsion stability, of phosvitin were much superior to those of bovine serum albumin which is an excellent emulsifier. The emulsifying activity and emulsion stability of phosvitin were greatly decreased by the partial removal of phosphate with phosphatase and by the complete removal of phosphate with alkaline treatment. In addition, the emulsifying properties were decreased by the blocking of phosphate in phosvitin with calcium ion. These results suggest that the electrostatic repulsive force of phosphate in phosvitin significantly affects its emulsifying properties.     

Occurrence of a large molecular size form of polyphosphate-glucose phosphotransferase in extracts of Mycobacterium tuberculosis H37Ra

Extracts prepared by sonicating Mycobacterium tuberculosis H37Ra cells with subsequent centrifugation at 18,000 X g proved to contain a very large molecular size form of polyphosphate-glucose phosphotransferase. The enzyme was separable by polyacrylamide gel electrophoresis, DEAE-cellulose chromatography or ultracentrifugation. When rechromatographed at alkaline pH values, it gave rise to one of the soluble forms of lower molecular weight. The conversion also took place as a result of n-butanol extraction or salting out with ammonium sulfate and heating of dissolved pellet. Under certain conditions the lower-molecular weight enzyme converted to the higher-molecular weight form by association with a hitherto undefined cell constituent. It is assumed that both ionic and hydrophobic forces play a role in this interconversion phenomenon.     

The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]

After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.     

雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究

利用盐析,离子交换,疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094u/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32KDa。性质分析表明：该蛋白酶在60℃、pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0～9.0的范围有很好的稳定性;1mM的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸（尤其是亮氨酸）构成的肽键有很强的水解能力。     

Pea,Chickpea and Lentil Protein Isolates: Physicochemical Characterization and Emulsifying Properties

This work is focused on physicochemical and emulsifying properties of pea (PP), chickpea (CP) and lentil (LP) proteins. We evaluated the molecular weight distributions, surface net charge, free sulfhydryl group (SH) and disulfide bond (SS) contents, protein solubility and thermal stability of the protein isolates. Their emulsifying properties (droplet size distribution, flocculation, coalescence and creaming) were also determined as function of pH values. The three protein isolates exhibit similar physicochemical properties, including good solubility and high thermal stability despite a high degree of denaturation. In addition, we analysed the influence of pH on stability of oil-in-water (O/W; 10 wt%/90 wt%) emulsions stabilized by the legume protein isolates. Concerning emulsifying ability and stability, the most unfavourable results for all three protein isolates relate to their isoelectric point (pI?=?4.5). A significant improvement in emulsion stability takes place as the pH value departs from the pI. Overall, this study indicates that pea, chickpea and lentil proteins have great potential as food emulsifiers.     

Size and stability to sodium dodecyl sulfate of alkaline phosphatases from their three established human genes

Subunit molecular weights of human alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) were dependent upon acrylamide concentration, a reflection of their glycoprotein nature. Molecular weights at a concentration of 7% (w/w) or greater were 68300, 80800 and 79400 for the enzymes from placenta, liver and mucosa of small intestine, respectively. All enzymes were dimers, the respective native Mr values determined by gradient gel electrophoresis being 138000, 186000 and 180000. None of the molecular weights was altered by desialylation. Stability of the catalytic activity of the purified enzymes to SDS varied and was very dependent on pH. SDS at 1% (w/v) rapidly denatured both native and desialylated alkaline phosphatase from placenta at pH 7.5 but had little effect on these at pH 10.3. Compared with placenta, the native enzyme from liver had greater stability at pH 7.5 and both native and desialylated forms had lower stability at pH 10.3. The enzyme from intestinal mucosa was sharply different from the other two isoenzymes: SDS had little effect at pH 7.5 but very rapidly denatured the enzyme at pH 10.3. The size of alkaline phosphatases and their stability to SDS can be used to identify gene products and to recognize heterodimers formed between products of more than one gene.     

Lipase-catalyzed hydrolysis of fish oil in an optimum emulsion system

In this study, fish oil was hydrolyzed by lipase in a fish oil-in-water emulsion system in an effort to improve the functional properties of fish oil. Lipase activity was found to depend on the quality of the water/fish oil interface area. We selected several suitable emulsifiers, and their emulsifying activities were evaluated under a variety of conditions, including concentration, water-oil ratios, pH values, and temperature. Among the selected emulsifiers, the emulsifying activity of gelatin was higher than those of carboxymethyl chitin (CM-chitin), bovine serum albumin, and Tween-20, all of which are commercial emulsificers Moreover, the emulsifying activity of the gelatin solution was the highest at 0.5%, and was reduced with increasing concentrations of above 1%. The optimal water-oil ratio, pH, and temperature conditions were 40% (w/v), pH 8.0 and 40°C, respectively. Under these conditions, the emulsifying activity of gelatin solution was 86%. The emulsion structure of the gelatin solution was characterized by high density and small particle size. The degree of sardine oil hydrolysis in the emulsion system was 50% higher than that of the non-emulsion system. The lipid species of the lipase-prepared sardine oil hydrolysates were identified as triacylglycerol, 1,3- and 1,2-diacylglycerol, monoacylglycerol, and fatty acid.     

Heat Denaturation and Emulsifying Properties of Plasma Protein

The effects of pH and NaCl on the denaturation of plasma protein during heat treatment were investigated, as well as the relationship between protein structure and emulsifying properties. When the plasma protein solution (1% w/v) was heated at 80°C, precipitation was accelerated by the presence of NaCl. The measurement of SH groups, surface hydrophobicity and CD spectrum revealed that denaturation occurs easily by heat treatment in the neutral pH region and in the presence of NaCl. The emulsifying activity index (EAI) did not change much after heat treatment at pH 3 irrespective of the presence of NaCl, but it decreased about 60% after heat treatment at pH 7 in the absence of NaCl. Gel filtration patterns indicated that a high molecular weight peak arose upon heat treatment at neutral pH. We concluded that the decrease in EAI was owing to the polymerization of serum albumin and γ-globulin, which are the main components of plasma protein, and disulfide bonds participated in this process.     

Molecular nature of three liver alkaline phosphatases detected by drug administration in vivo: differences between soluble and membranous enzymes

1. Activities of alkaline phosphatase, liver-membranous, liver-soluble and serum-soluble, were dramatically induced in dogs by treatment with both phenobarbital and brovanexine. The treatment induced a 17-fold increase in membranous, a 155-fold increase in soluble, and a 105-fold increase in serum alkaline phosphatases. 2. There was no difference in the enzymatic behavior of the three forms of alkaline phosphatase, on heat stability, amino acid inhibition and optimum pH. 3. When the three alkaline phosphatases were treated initially with n-butanol, their apparent molecular size was identical. After treatment with phosphatidylinositol-specific phospholipase C, the liver-soluble and serum-soluble alkaline phosphatase were of the same molecular size. Liver-membranous alkaline phosphatase, however, was larger in molecular size than the other two forms, suggesting a difference between soluble and membranous alkaline phosphatase forms. 4. In terms of the sugar moiety of the three alkaline phosphatase forms, the membranous enzyme showed more of the higher affinity fraction and less of the lower affinity fraction of concanavalin A, compared with the soluble enzymes. 5. Consequently, it is possible that the membranous enzyme may be solubilized by an enzyme such as phosphatidylinositol-specific phospholipase C and modify further the sugar moiety of alkaline phosphatase molecules, resulting in serum alkaline phosphatase transfer from the soluble enzyme in liver.     

Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating

Hyaluronan (hyaluronic acid, HA) was depolymerised by ultrasonication (US), microwave irradiation (MW) and conventional heating (CH), and the effect of pH and oxidants was investigated. The degradation was followed by viscometry and size exclusion chromatography coupled with low-angle light scattering. The results demonstrated that depolymerisation of HA by US leveled off to a limiting molecular mass, and the degradation was significantly enhanced by acidic and alkaline pH only in the presence of oxidants. In contrast to US, the course of depolymerisation by MW was strongly pH-dependent, and the degradation rate increased with decreasing pH. The expected enhancement of depolymerisation by MW in comparison to CH was marked only at very short heating time at pH <4. The NMR and FTIR spectral analyses indicated that HA in the whole M_w-range studied retained almost the backbone of the parent polysaccharide independently on the degradation method used. At harsh degradation conditions (long-term treatments, particularly at acidic pH or alkaline pH and in presence of oxidants) the depolymerisation was accompanied by destruction of both constituent sugar residues and formation of unsaturated structures detectable by UV-absorption at 230–240 and 260–270 nm. US-assisted oxidative degradation under mild reaction conditions was shown to be the most appropriate procedure to reduce the molecular mass of HA to 100 kDa without significant chemical modification of the polysaccharide.     

Some properties of the lysozymes in serum and colostrum from cows with high and low lytic power against Micrococcus lysodeikticus

Previous work has uncovered a dominant gene for high bacteriolytic activity of bovine serum against the test bacterium Micrococcus lysodeikticus. This major gene effect is also fully expressed in colostrum. In the present study the lytic power of serum and colostral whey from high and low level cows was subjected to a degree of characterization. It was found that the enzyme activities studied exhibited properties in accordance with those defined for a lysozyme (EC 3.2.1.17), i.e. (1) lysis of a suspension of M. lysodeikticus, (2) basic protein (pI = 10.0 and pI = 10.3 for bovine serum lysozyme (BSL) and bovine colostrum lysozyme (BCL), respectively), (3) molecular weight (MW) approximately 16 000 for both BSL and BCL, (4) liberation of free reducing sugars during action on cell wall peptidoglycan (the kinetics of BSL and BCL differed strongly), and (5) fairly heat stable, especially at acidic pH and relative labile at alkaline pH (BCL was far more sensitive to heating at alkaline pH than was BSL). The dramatic differences in activity between high and low level animals might be due to a major genetic mechanism influencing the amount of, or the activity of, circulating enzyme molecules, rather than a structural gene coding for a certain enzyme with a particular specific activity. This is also supported by the high correlation between the lytic capacity of BSL and BCL in spite of the different properties of these lysozymes (i.e. in respect of pI, enzyme kinetics and heat stability) reported in the present study.     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

Scrapie PrP 27-30 is a sialoglycoprotein.

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.     

Isolation and Identification of 6-Methylsalcylic Acid and Gentisyl Alcohol from Phyllosticta sp.

Bovine serum albumin was reduced by incubating with various concentrations (0–200 mM) of 2-mercaptoethanol, and its emulsifying properties were examined for an oil-in-water emulsion system. A particle size analysis revealed that albumin reduced at 30 mM of the thiol yielded smaller oil particles than either native protein, or the protein reduced at 70 or 200 mM of the thiol. Furthermore, the particle size was almost constant during 35 days of storage with albumin reduced at 30 mM of the thiol, while an emulsion prepared using the native protein, or the protein reduced at 70 or 200 mM of the thiol was unstable during the same storage period. Gel filtration chromatography and transmission electron micrography show that serum albumin made aggregates with high molecular size by its disulfide reduction with 70 or 200 mM, but not with 30 mM of 2-mercaptoethanol. It was, therefore, concluded that the emulsifying property of serum albumin can be improved by a mild disufide reduction.     

Alasan, a new bioemulsifier from Acinetobacter radioresistens.

Acinetobacter radioresistens KA53, isolated by enrichment culture, was found to produce an extracellular, nondialyzable emulsifying agent (referred to as alasan) when grown on ethanol medium in a batch-fed reactor. The crude emulsifier was concentrated from the cell-free culture fluid by ammonium sulfate precipitation to yield 2.2 g of emulsifier per liter. Alasan stabilized a variety of oil-in-water emulsions, including n-alkanes with chain lengths of 10 or higher, alkyl aromatics, liquid paraffin, soybean and coconut oils, and crude oil. Alasan was 2.5 to 3.0 times more active after being heated at 100 degrees C under neutral or alkaline conditions. Emulsifying activity was observed over the entire pH range studied (pH 3.3 to 9.2), with a clear maximum at pH 5.0. Magnesium ions stimulated the activity both below (pH 3.3 to 4.5) and above (pH 5.5 to 9.3) the pH optimum. Alasan activity was higher in 20 mM citrate than in 20 mM acetate or Tris-HCl buffer. Preliminary chemical characterization of alasan indicated that it is a complex of an anionic, high-molecular-weight, alanine-containing heteropolysaccharide and protein.     

Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.

The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.     

Lysozyme-Sensitive Bioemulsifier for Immiscible Organophosphorus Pesticides

Two Bacillus strains capable of emulsifying immiscible organophosphorus pesticides were isolated by enrichment methods. The emulsifying factor produced by Bacillus strain FE-2 has a high molecular weight, is Iysozyme sensitive and thermostable, and can be precipitated with trichloroacetic acid or ammonium sulfate; it may be a glycolipopeptide. It is specific for immiscible organophosphorus pesticides and is secreted during growth in the presence of such pesticides.     

Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

The lipolytic activities of heart tissue towards full and partial acylglycerols were characterized. Tissue lysosomal, acid lipase activity (pH 4.8) was inhibited by high salt, protamine sulfate, NaF, MgATP, Triton X-100, serum and the esterase-inhibitor diethylparanitrophenyl phosphate. The tissue neutral triacylglycerol lipase activity (pH 7.4) was recovered predominantly in the microsomal and soluble fractions and exhibited essentially identical properties towards activators (serum, apolipoprotein C-II) and reagents (NaCl, Triton X-100, NaF, MgATP and diethylparanitrophenyl phosphate) relative to vascular lipoprotein lipase, except for protamine sulfate which increased the serum-stimulated neutral triacylglycerol lipase activity. Triacylglycerol hydrolysis at acid pH was incomplete, whereas at neutral pH full hydrolysis occurred. Myocardial mono- and diacylglycerol lipase activities, with pH optima of 8.0 and 7.4, respectively, were recovered in the microsomal fraction. They differed immunologically from neutral lipase and lipoprotein lipase and did not bind to heparin-Sepharose 4B. They were kinetically different, partially inhibited by NaCl and differentially affected by protamine sulfate. NaF, Triton X-100 and diethylparanitrophenyl phosphate. Our data suggest that endogenous hydrolytic activity against full and partial acylglycerols is mediated by separate enzymes.     

Isolation and partial characterization of a type II Fe receptor from a group A streptococcus

A group A streptococcal strain rich in Fc receptors was selected by an immunoblotting technique and used as the source for isolation of a functionally active Fc receptor. A variety of extraction techniques were compared including (1) heat extraction at neutral, acid or alkaline pH, (2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin, papain or phage lysin, or (3) autoclaving or heating in the presence of sodium dodecyl sulfate. The most homogeneous receptor was recovered following heat extraction and contained two molecular weight forms. The major form had a molecular weight of 56 000 daltons and the minor form had a molecular weight of 38 000 daltons. These two proteins could be isolated without loss of activity by binding to and elution from a column of immobilized human IgG. An antibody prepared against a single form of the affinity purified receptor demonstrated reactivity with both molecular weight forms of the heat extracted receptor. The group A receptor was found to be both antigenically and physicochemically distinct from either the type I receptor found on the majority of Staphylococcus aureus strains or the type III Fc receptors found on the majority of group C streptococcal strains.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.