SDS-polyacrylamide Disc Gel Electrophoresis Research Articles

Soluble proteins of guinea pig seminal vesicle lumen: purification and partial characterization.

Luminal contents of guinea pig seminal vesicle were extracted with 0.154 M NaCl and the soluble protein fraction was studied by DEAE-cellulose and Sephadex G-100 column chromatography; anionic, cationic, and SDS-polyarcylamide disc gel electrophoresis; isoelectric focusing; molecular weight determinations by SDS-polyacrylamide disc gel electrophoresis; analysis for neutral carbohydrate; sucrose density-gradient centrifugation for determination of sedimentation coefficients; and amino acid analyses. Four protein fractions (referred to as proteins 1, 2, 3, and 4 in order to elution) were obtained by DEAE-cellulose column chromatography. Proteins 1, 2, and 4 gave single major bands in multiple disc gel electrophoretic systems. Protein 3 gave one major band and one minor component believed to be a fragment of a primary vesicular protein. Protein 1 is very basic and probably represents the clotting protein of guinea pig semen. The three nonclotting proteins could not be detected in serum and are probably intrinsic to seminal vesicle epithelium. All four proteins were found in animals weighing 700 g or more; only two of the three nonclotting proteins were found in more than half of the younger animals. This makes it even more likely that one or more of the nonclotting proteinc could serve as a useful gene marker in studies of androgen mechanisms.

Purification and characterization of a levanase from Streptomyces sp. 366L

A levanase-producing microorganism was isolated from soil and identified as genus Streptomyces. The enzyme was purified to homogeneity by several procedures including ammonium sulfate fractionation, DEAE-Toyopearl 650M ion exchange chromatography, phenyl-Toyopearl 650M hydrophobic chromatography, Sephadex G-200 gel filtration, and hydroxyapatite adsorption chromatography. The molecular mass of the enzyme was estimated to be 78 000 by SDS-polyacrylamide disc gel electrophoresis and 80 000 by gel filtration. The isoelectric point of the enzyme was pH 4.1 and activity was optimal at pH 7.0 and 40°C. In 30 min reactions, the enzyme was stable at the pH range of 6.0–10.0 at 20°C and remained stable up to 45°C at pH 7.0. The enzyme hydrolyzed levan to produce levanheptaose predominantly and showed an absolute substrate specificity for levan. When incubated with levans from Serratia sp. and Zymomonas mobilis, the enzyme hydrolyzed about 81 and 61%, respectively.

Thermostable phenylalanine dehydrogenase from a mesophilic microbacterium sp. strain DM 86-1

Bacteria that produced NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (Mr) of approximately 330,000. The enzyme is composed of eight identical subunits with an Mr of approximately 41,000. The apparent Km values for l-phenylalanine and NAD+ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55 degrees C for 10 min.

Inhibition of ileal brush-border chloride conductance by specific antibody.

Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (> 110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass > 100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

Studies on restriction endonucleases of acetic acid bacteria and allied organisms. Part VI. Purification, properties and determinations of recognition sequence and cleavage site of restriction endonuclease from "Agrobacterium gelatinovorum" IAM 12617, a marine bacterium (AgeI).

A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, "Agrobacterium gelatinovorum" IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved lambda and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, phi X174 RF I, M13mp 18 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37 degrees C and pH 7.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 50 mM NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5'-ACCGGT-3' and cut between A and C, producing a 5'-cohesive tetranucleotide extension.

Purification of glucose 6-phosphate dehydrogenase from the sea-urchin, Hemicentrotus pulcherrimus

1. 1. Glucose 6-phosphate dehydrogenase (G6PD) was purified from ovaries of the sea-urchin, Hemicentrotus pulcherrimus, by the sequential column chromatography of Ultrogel AcA 34, Blue-Sepharose CL6B, DEAE-Sephadex and 2′,5′-ADP-Sepharose 4B. 2. 2. The enzyme thus purified showed the specific activity of 267 with 20% yield and was proved to be homogeneous by SDS-polyacrylamide disc gel electrophoresis. 3. 3. The purification procedure used in this study has several advantages and was effective generally in G6PDs of other sea-urchin species.

The effect of l-carnitine on the apolipoprotein pattern of rats fed a cholesterol-rich diet

1. 1. l-Carnitine was administered to hypercholesterolemic rats for eight days, after which the blood was collected by intracardiac puncture and lipoproteins were collected by preparative ultracentrifuge. 2. 2. The chemical composition of lipoproteins of normal rats and hypercholesterolemic rats before and after l-carnitine treatment was determined. 3. 3. The total serum B and E apoproteins were assayed by rocket electrophoresis, while apoproteins in VLDL, IDL, LDL and HDL were determined by densitometric scanning of SDS and TMU polyacrylamide disc gel electrophoresis. 4. 4. The results obtained showed that the l-carnitine treatment tends to restore to normal values both the chemical composition of lipoproteins and the apoproteins pattern of rats fed on a diet enriched with cholesterol.

Differential racemization of aspartate and serine in human myelin basic protein

L-Aspartate and L-serine were found to undergo amino acid racemization in brain myelin basic protein (MBP) of aging humans. The observed racemization was different in each chromatographically purified MBP isoform. Pepsin digestion of MBP produced three peptides, each of which exhibited different degrees of racemization of the same amino acids. MBP isolated by the same method from rat and guinea pig brain showed little accumulation of D-amino acids. Total MBP isolated from SDS-polyacrylamide disc gel electrophoresis of total human myelin proteins (delipidated myelin) was racemized to the same extent as purified MBP, indicating that the racemization observed was not an artifact of the isolation procedure. Myelin proteolipid protein from the same gel was racemized approximately half as much as MBP. The age and environment of the aspartates and serines in myelin proteins may strongly affect their observed racemizations.

1,2-α- D-Mannosidase from a wood-rotting basidiomycete, Pycnoporus sanguineus

An acidic α- D-mannosidase has been isolated from the culture filtrate of a wood-rotting Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme determined. The extracellular mannosidase was homogeneous on PAGE at pH 9.4. The M r as determined by SDS-polyacrylamide disc gel electrophoresis was 64000, and the pI was pH 4.7 using electrofocusing. The purified enzyme had a pH optimum of 4.5 with Baker's yeast mannan and had no activity towards p-nitrophenyl-α-mannoside. The K m and k cat values for Manα1-2Man at pH 4.5 and 30° were 0.9 mM and 1.9 sec. the enzyme had no activity towards Manα1-3Manα1-2Man, and it cleaved specifically the 1,2-α-linked side chain of yeast α-mannan, producing free α- D-mannose.

The biosynthesis of nicotinamide adenine dinucleotide. Part XVIII. Purification and some properties of nicotinate phosphoribosyltransferase from hog liver.

Purification of nicotinate phosphoribosyltransferase (NPRTase, EC 2.4.2.11) from hog liver and some of its properties were investigated. Purified enzyme was found to be homogeneous by the criterion of polyacrylamide gel disc electrophoresis and to have a molecular weight of about 120,000. The subunit molecular weight was found to be 64,000, using SDS polyacrylamide gel disc electrophoresis. The optimum pH and isoelectric point of the enzyme were 7.3~7.4 and 4.8, respectively. Mn2+, Co2+, and Mg2+ were effective in meeting the NPRTase requirement for a divalent cation. ATP had a stimulative effect on the enzyme activity in the presence of Mg2+, on the other hand, in the presence of Mn2+, ATP had an inhibitory effect on the enzyme activity. Of the purine nucleotides examined, only ATP stimulated NPRTase activity, and GTP did not. On the contrary, all other nucleotides inhibited the activity. Nicotinate mononucleotide, which is a reaction product, inhibited NPRTase activity. Among the nicotinic acid analogue...

Purification and characterization of aminopeptidase from Euphausia superba.

Krill aminopeptidase was purified about, 1, 100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140, 000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg++ and amastatin, and partially by bestatin, and was activated by Co++. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast. These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.

Postnatal development of rat kidney plasma membrane Na-K-ATPase.

The postnatal development of the sodium transport mechanism in the rat kidney, as judged by the activity of renal plasma membrane Na-K-ATPase, was studied. Highly purified kidney plasma membranes as verified by electron microscopic and enzyme examinations, were used. Na-K-ATPase activity (mumol Pi/mg protein/h) increases exponentially from 16.9 +/- 1.94 found at birth to the mature level of 43.1 +/- 2.16 reached at the age of 41 days. This finding paralleled our results of SDS-polyacrylamide gel disc electrophoresis, showing a completely differentiated plasma membrane protein structure at birth, the maturation of which proceeds only as quantitative enrichment of some protein fractions in the further postnatal period.

Action of Levan Fructotransferase of <italic>Arthrobacter ureafaciens</italic> on Levanoligosaccharides

Levan fructotransferase of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular transfructosylation reaction, was purified to give a single protein band of pI 4.5-4.7 on isoelectric focusing. It had a molecular weight of 128,000 on gel-filtration on Sephadex G-200 and 60,000 on SDS-polyacrylamide disc gel-electrophoresis, suggesting that the enzyme is composed of two identical subunits. The shortest levanoligosaccharide chain required for the difructose anhydride IV formation was determined to be tetraose. TLC of the enzymic digest of a modified levanhexaose derived from levanhexaose by the reduction of the reducing end to an alditol residue with sodium borohydride gave the difructose anhydride IV spot, suggesting that the enzyme attacks the modified levanhexaose molecule from the direction of the non-reducing fructose end. The enzymic digests of levantetraose, -pentaose, and -hexaose as the substrate gave, in addition to the difructose anhydride IV spot, spots of oligofructans of lower mobility than the original substrate on TLC. From the digest of levantetraose, a hexaoligofructan and a smaller amount of a pentaoligofructan but no fructose were separated, indicating enzymic intermolecular levanbiosyl and fructosyl transfer reactions.

Electrophoretic Studies of the Seed Proteins of Cowpea, Vigna unguiculata (L.) Walp.

Summary The globulin fraction accounted for the major proportion (72%) of the protein extracted from mature cotyledons of cowpea, Vigna unguiculata (L.) WALP. cv. Vita 3. The globulin and albumin fractions were compared by pore-gradient polyacrylamide gel electrophoresis, and component polypeptides were analysed by SDS polyacrylamide disc gel electrophoresis. The major globulin, vignin, consists mainly of non-covalent associations of polypeptides of MW 56,000 and 52,500 daltons. Under reducing conditions, globulin polypeptides of MW 80,000 and 43,000 dissociate and polypeptides of 61,000, 26,500 and 19,000 daltons are formed. Under the same conditions, the major polypeptides of the albumin fraction are distinct from those of the globulin fraction, having sizes estimated to be ca. 100,000, 70,000, 43,000, 35,000, 32,000 and 25,000 daltons. During embryo development there is no clear division into a period of albumin synthesis followed by a period of globulin synthesis; polypeptides representing both fractions were present from the earliest stage analysed, 7 days after anthesis. There are, however, differences in the periods of most rapid net synthesis for individual polypeptides of each fraction.

赤血球膜蛋白への血清脂質とリポ蛋白の関与

The impairment of blood fluidity facilitates the formation of ischemic lesion with respect to decrease in blood supply to tissues under the participation of changes in arterial wall and local blood pressure gradient. Functional compensation due to augmentation of blood fluidity for decrease in blood supply to tissues would protect even elderly adults with systemic arteriosclerotic changes from the occurrence of thrombotic diseases. Blood fluidity is influenced by corpuscular and plasma components in the blood. Erythrocytes participate to low blood fluidity by increasing viscosity and decreasing the deformability induced by low stroma ATP content and quality changes in membrane constituents affected by plasma lipids and lipoproteins. This study was intended to clarify fundamental association of serum lipids with erythrocyte membrane proteins as a part of lipid and hemorheological research in thrombotic cerebrovascular disorders.Seventy-nine healthy adults with normal physical findings, blood pressures, urinalysis, blood sedimentation rate, chest films, ECG, liver function tests and serum lipids were selected for this study in the annual screening for circulatory disease. Blood specimens were drawn in the fasting state early in the morning. Erythrocyte membrane was separated by the method of Dodge et al., the membrane proteins were extracted and analyzed qualitatively using SDS-polyacrylamide gel disc electrophoresis by the method of Weber et al., Total cholesterol (TCh), triglyceride and phospholipid were determined by conventional methods, HDL-cholesterol (HDL-Ch) by MgCl2-dextran sulfate method, LDL and VLDL by the precipitating method, and apo A-I, apo A-II and apo B by single radial immunodiffusion method.Subjects aged above 60 years revealed significantly higher levels of TCh, LDL and apo B, and significantly lower levels of HDL-Ch and HDL-Ch/TCh ratio compared with those aged 20 to 39 years. Erythrocyte membrane band 2.1 connecting with spectrin affecting immediately erythrocyte deformobility was lower in subjects aged above 60 years than in those aged 20 to 39 years. In subjects aged 20 to 39 years, serum TCh, esterified Ch and LDL were directly proportional to spectrin 1, spectrin 2 and inversely to glucose 3 phosphate dehydrogenase, and HDL-Ch directly to band 4.1, 4.2, 8 and actin (Table 3). LDL was directly proportional to spectrin (band 1+2) (Fig. 2). In subjects aged 40 to 59 years, apo A-I was inversely proportional to actin, apo A-II directly to band 4.1 (Table 4). In subjects aged above 60 years, A-I was directly proportional to band 2.1 (Fig. 3). Neither of age groups showed significant relationships between serum unesterified Ch and membrane proteins.Erythrocyte lipids are almost located in the membrane. The mature erythrocyte is incapable of the denovo synthesis of cholesterol. A notable feature of blood cholesterol is its ability to exchange between erythrocyte and plasma. Morphological observations have shown the appearance of various kinds of abnormal erythrocyte, which impaires hemorheological behavior following the changes of membrane lipids with regard to the membrane protein and stroma ATP. Although membrane lipids analysis is lacking in our study, intimate associations of serum lipids and lipoproteins with the membrane protein were shown. Therefore serum lipids should be regarded as one of important factors in the development of hemorheological disorders as well as the participation in atherosclerosis in thrombotic disorders.

Isolation and Characterization of Nucleases from a Clinical Isolate of Serratia marcescens kums 39581

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).

Immunochemical investigations of Rh0(D) activity detected in Band 3 of red cell membrane.

Stroma from D positive red cell was solubilized with 1% deoxycholic acid (DOC) and was applied onto Sepharose 4B and 6B, followed by fractionation with activated thiol Sepharose 4B. By this method Band 3 containing no carbohydrate was prepared. The Band 3 was incubated with human anti-D, then immune complex soluble in DOC solution was produced. This complex was not prepared from red cell components other than Band 3, nor from Band 3 from D negative red cell. The complex agglutinated papainized D positive red cells. Molecular weight of the complex was estimated to be 500,000 to 700,000 by the gel filtration method. The molecular weight increased according to the time of incubation. The complex gave a precipitation line in counter immunoelectrophoresis (IEP), a single peak in crossed IEP against anti-Band 3, and a line at gamma-globulin region in IEP against anti-human serum. SDS-polyacrylamide gel disc electrophoresis of the complex showed the pattern consisting of fragments of immunoglobulin G and Band 3 which was not stained with periodic acid Schiff reagent. These support our results that Rh-Hr blood type activities are located in Band 3 almost free of carbohydrate.

Association of spermine and 4S RNA during axonal transport in regenerating optic nerves of goldfish

Experiments were designed to determine whether polymines are bound to 4S RNA and then transported axonally along regenerating optic axons of goldfish. In one set of experiments, inhibition of retinal RNA synthesis by intraocular injections of 10 μg of cordycepin, blocked the axonal transport of both [ 3H]RNA and [ 14C]spermidine by about 65%, 6 and 14 days after injection. Intraocular injections of vinblastine, (0.1, 0.5 or 1.0 μg) an agent which interrupts axonal transport of proteins, had no effect on retinal RNA synthesis nor on the amount of [ 14C]spermidine incorporated into the TCA-insoluble fraction of retinal extracts. However, the axonal transport of both [ 3H]RNA and [ 14C]polyamines was affected in a dose-dependent fashion; the inhibition of both was approximately 80% at the higher dose. Further evidence for an association between axonally transported 4S RNA and polyamines came from experiments in which regenerating optic axons were cut and allowed to degenerate 6 days after injection of [ 3H]spermidine into the eye. The loss of optic axons from the tectum 7 days after cutting the nerve resulted in an 86% loss of TCA insoluble polyamines, indicating a largely intra-axonal locus. A similar loss of 4S RNA was found in identical experiments following injections of [ 3H]uridine into the eye. Finally, experiments were performed in which [ 3H]spermidine was injected into both eyes of 12 fish whose optic nerves had been regenerating for 18 days. Six days later, fish were sacrificed and RNA was extracted from tectal homogenates by hot phenol and ethanol precipitation. The major stable RNA species were separated by SDS-polyacrylamide disc gel electrophoresis and radioactivity was determined by extraction of 2.0 mm gel slices. Results showed co-migration of 3H with 4S RNA optical density peaks, and not with 28S and 18S ribosomal RNA peaks, suggesting that some polyamine-associated radioactivity is bound to axonally transported 4S RNA. When the nature of that radioactivity was determined on an amino acid analyzer, it was found to be present primarily as spermine and not as the injected compound spermidine. The data are consistent with the hypothesis that some spermine is bound to 4S RNA and then axonally transported along regenerating axons of the goldfish optic nerve.

Fatty acid synthetase in human adipose tissue: Purification and characterization

1. 1. Fatty acid synthetase of abdominal subcutaneous adipose tissue of humans obtained at autopsy was purified 95 times with a final recovery of 10%. 2. 2. The purity of the enzyme was verified by polyacrylamide disc gel electrophoresis and isoelectric point determination. Both techniques revealed only one protein band. 3. 3. The fatty acid synthetase molecular weight was determined by SDS-polyacrylamide disc gel electrophoresis and found to be 500,000. 4. 4. The fatty acid synthetase K m for both NADPH and malonyl-CoA were also determined and were found to be 18 and 28 μM respectively. 5. 5. The presence of an active fatty acid synthetase in human adipose tissue raises the obvious question as to its biological role.

Isolation of heart- and kidney-binding protein from group A streptococci.

Tritium-labeled, water-soluble components of Streptococcus pyogenes type M6 absorbed to cardiac tissue in vitro. Tissue binding was time dependent, saturable, and reversible. Chromatography of the crude bacterial extract on Bio-Gel P-300 indicated a molecular weight greater than 300,000 for the heart-binding component. Sodium dodecyl sulfate (SDS) dissociated this aggregate into a protein of 18,000 to 20,000 daltons as determined by Sephacryl S-200 chromatography and SDS-polyacrylamide disc gel electrophoresis. The tissue-binding protein was also purified from streptococcal extracts by absorption to immobilized heart components. SDS-polyacrylamide gel electrophoresis of the protein desorbed from tissue revealed a radioactive band of 19,000 daltons. Indirect immunofluorescence tests on cardiac tissue treated with streptococcal extract showed an accumulation of a bacterial antigen on the sarcolemmal sheaths. Streptococcal components also adsorbed to basement membranes of kidney. Antisera prepared to isolated cytoplasmic membranes and water-soluble extracts of S. pyogenes type M6 were the most sensitive reagents for the detection of bacterial components bound to tissue. Antisera prepared to isolated cell walls and to intact bacteria were weakly reactive in these assays.