Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Subunit structure of -acetohydroxy acid isomeroreductase from Salmonella typhimurium

α-Acetohydroxy acid isomeroreductase, purified from Salmonella typhimurium, has a molecular weight of 220,000. The native enzyme consists of a tetramer of four identical subunits on the basis of the following criteria: (1) SDS gel electrophoresis revealed a single component of molecular weight 55,000 (2) carboxypeptidase digestion of the enzyme revealed 4 moles of glycine released per mole of enzyme; (3) amino acid analysis of the native enzyme indicated 204 moles of lysine and arginine; (4) after tryptic digestion, a total of 51 peptides were detected by high voltage electrophoresis and descending chromatography. In the native enzyme, it was possible to tititrate 8 sulfhydryl groups per mole of enzyme. Neither the rate nor extent of sulfhydryl titration was affected by substrates or products. After denaturation with SDS or urea, 8 additional sulfhydryls per mole of enzyme were titrated.     

The antitumor and antioxidative activities of polysaccharides isolated from Isaria farinosa B05

The water-soluble intra-polysaccharides WIPS1 and water-soluble extra-polysaccharides WEPS1 were isolated from Isaria farinosa B05 through ethanol precipitation and gel permeation chromatography (GPC). Their characteristics were determined by chemical analysis, gas chromatography, GPC and IR spectroscopy. The results show that WIPS1 contained 90.3% carbohydrate, 8.00% uronic acid, 7.15% protein and three kinds of monosaccharides including mannose, galactose and glucose with a molar ratio of 8.0:4.8:1.0. WEPS1 contained 93.4% carbohydrate, 8.06% uronic acid, 4.40% protein and three kinds of monosaccharides including mannose, galactose and glucose with a molar ratio of 21.6:4.7:1.0. WIPS1 and WEPS1 had a molecular weight of 42 and 208kDa, respectively. The in vivo tests in mice indicate that WIPS1 and WEPS1 had significant antitumor and antioxidative activities to some extent.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH₂-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

Isolation and characterization of fin whale prolactin.

A highly purified prolactin preparation has been obtained from fin whale pituitaries by extraction with acid acetone. salt precipitation, isoelectric fractionation, and exclusion chromatography on Sephadex G-100 and ion-exchange chromatography on DEAE-CELLULOSE. Fin whale prolactin was isolated in a yield of 250 mg/kg wet weight tissue. It was found to have a molecular weight (SDS disc gel electrophoresis) of 23,600 daltons and an alpha-helix content (circular dichroism) of 50%. The amino acid composition and circular dichroism spectra were very similar to those of porcine prolactin. The partial amino acid sequence has been determined by the method of fluorescein-isothiocyanate. Fin whale prolactin was found to be 80% as potent as ovine prolactin with regard to pigeon crop-sac assay.     

Isolation and characterization of a soybean lectin having 4-O-methylglucuronic acid specificity

A new lectin from soybeans having specificity toward the extracellular 4-O-methyl-D-glucurono-L-rhamnans produced by certain strains of Rhizobium japonicum has been purified and characterized. Isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose. Residual globulins were removed by adsorption of the lectin on concanavalin A-agarose and elution with methyl alpha-mannoside. The lectin is a glycoprotein (3-5% carbohydrate) with a molecular weight of approximately 175 000. It is a tetramer with subunit molecular weights of 45 000 when dissociated with sodium dodecyl sulfate. Reverse-phase high-pressure liquid chromatography analysis indicates the presence of two types of subunits, both having equivalent molecular weights. According to amino acid analyses, the lectin is rich in acidic but low in sulfur-containing amino acids. The carbohydrate portion of the lectin contains mannose; no hexosamines could be detected. Chemical modification of the lectin indicated that neither sulfhydryl groups nor amino groups participate in binding. Quantitative binding studies of the lectin with various carbohydrate haptens showed that specificity was directed toward 4-O-methyl-D-glucuronic acid, D-glucuronic acid, and their methyl glycosides with 4-O-methyl-D-glucuronic acid 3-4-fold more effective. In each instance, the methyl glycoside is a more effective hapten.     

Purification and Properties of the Periplasmic Glucose-Binding Protein of Pseudomonas aeruginosa

A glucose-binding glycoprotein (GBP) from the periplasm of Pseudomonas aeruginosa was purified to homogeneity as judged by polyacrylamide gel electrophoresis, molecular sieve chromatography, and double-diffusion gel precipitation. It had an average molecular weight of 44,500 and an isoelectric point of 4.7. One mole of glucose was bound per mole of GBP with a dissociation constant of 0.35 muM. The binding of radioactive glucose by GBP was not significantly inhibited by 10-fold-higher concentrations of other carbohydrates; however, a number of related compounds were found to compete at 100-fold-higher concentrations. Amino acid analyses revealed predominant amounts of alanine, glutamate, and glycine and a low content of sulfur-containing amino acids. The carbohydrate moiety of GBP, comprising nearly 16% of the total weight, contained galactosamine, glucosamine, fucose, galactose, glucose, and mannose. A GBP-deficient mutant, strain MB723, was found to be defective in both membrane transport and glucose chemotaxis. Strain MB724, a revertant to GBP-positive phenotype, simultaneously recovered normal levels of both membrane functions.     

Glycoproteins and glycolipids of oxyntic cell microsomes II. Glycopeptides and glycolipids

Glycosylated compounds associated with the carbohydrate-rich tubular membrane system of the oxyntic cell were investigated. Two glycopeptide fractions, designated Peaks A and B, were isolated from pronase digests of bullfrog oxyntic cell microsomes. Molecular sieve chromatography and cellulose acetate electrophoresis revealed that, although somewhat heterogeneous, each peak was composed primarily of glycopeptides with similar molecular weights and net charge densities. Peak B glycopeptides had a mean molecular weight of about 6000 and contained 70% of the recovered carbohydrate in the following molar ratios: hexose, 1.00; $\text{N-}\text{acetylhexosamine}$, 0.71; fucose, 0.61; sialic acids, <0.03. Peak a glycopeptides were considerably larger (molecular weight approx. 100 000) and contained carbohydrates in molar ratios similar to those of Peak B. In both peaks galactose and $\text{N-}\text{acetylglucosamine}$, respectively, were the predominant hexose and amino sugar isomers.The glycolipid content of bullfrog oxyntic cell microsomes was assessed by qualitative and quantitative thin-layer chromatography. The most abundant glycolipids were monoglucosylceramides (0.098 mole/mole phospholipid) and monogalactosylceramides (0.046 mole/mole phospholipid). Small quantities of sulfatides and gangliosides were also present.A compilation of available data regarding the chemical composition of the microsomes revealed that these membranes resemble plasma membranes in having high molar ratios of cholesterol to phospholipid (approx. 1.0) and large quantities of carbohydrate (225 μg/mg protein). The possible significance of these compositional features in protecting the oxyntic cell is discussed.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Purification and characterization of acid phosphatase in rat liver lysosomal contents

Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.     

A rapid and simplified procedure for purification of a cellulase from Fusarium lini

An endo-beta-1,4-glucanase (EC 3.2.1.4) was obtained in high yields in purified form a culture filtrate of Fusarium lini by an extremely simple method. The method consists of precipitation of the culture filtrate with ammonium sulphate (290 g/L), followed by chromatography of the precipitated fraction on Biogel P-150. The purification is based on the unusual property of the enzyme being eluted after cytochrome C, even though it molecular weight is 2.8 x 10(4) (by SDS PAGE). The yield of pure enzyme was 6.8 mg/L culture broth. The homogeneity of the enzyme was established by ultracentrifugation, isoelectric focusing, and electrophoresis in polyacrylamide gels containing SDS. The enzyme was isoelectric at pH 8.3 and contained 2.9% carbohydrate. The K(m) value for carboxymethyl (CM) cellulose was 11.6 mg/mL. The enzyme showed high viscosity reducing activity towards CM cellulose but very low activity with Walseth cellulose and crystalline celluloses such as Avicel and cotton. The purified enzyme has activity towards xylan. The amino acid analysis showed a predominance of acidic and neutral amino acids and low contents of histidine, arginine, and methionine. One-half of the cysteine content was 11 residues/mol enzyme, and no free-SH group was detectable.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Pig liver monoamine oxidase: Studies on the subunit structure

The molecular weight of pig liver MAO has previously been shown to be about 115,000 with 1 mole of covalently bound FAD per mole of enzyme. Gel filtration of purified enzyme on Sepharose 4B in 6 m guanidine and 0.1 m mercaptoethanol (MCE) and analytical ultracentrifugation in 0.1% sodium dodecyl sulfate (SDS) and 0.1% MCE yielded molecular weights of 55,000 and 63,000, respectively. By polyacrylamide electrophoresis in 0.1% SDS + MCE one band of 60,000 MW appeared. These results seem to imply that the enzyme is composed of two subunits of which one carries the active site. If MCE was omitted during the gel electrophoresis two equally large bands of about 60,000 MW were formed. By using enzyme inhibited by [¹⁴C]pargyline, a MAO-inhibitor blocking the active site of the enzyme in a 1:1 molar ratio, it was found, however, that both bands contained pargyline. Furthermore, amino acid analyses yielded the same amino acid composition of the two bands. The results are interpreted that the enzyme is composed of two subunits of identical molecular size (about 60,000) of which only one contains the active site and that the enzyme preparation contained two forms of the enzyme presumably differing in the number of disulfide bonds.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Purification and properties of the thermostable acid protease of Penicillium duponti.

An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.     

Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.     

Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.