Staphylococcal Enterotoxin D

Purification of enterotoxin D from Staphylococcus aureus 494 was attempted by utilizing the following techniques: Concentration of bacterial culture supernatant with polyethylene glycol 20000, CM-cellulose column chromatography, Sephadex G-100 gel-filtration, chromatography on a DEAE cellulose column with concave gradient elution and gel-filtration with Sephadex G-50. The final preparation obtained gave a single precipitin line with anti-crude enterotoxin D serum and no precipitation with anti-enterotoxin A, B or C when it was tested by Ouchterlony's technique. It was capable of producing 100% emesis in monkeys at about 1.25 μg of protein per kg body weight. Antiserum prepared by injecting rabbits with the purified preparation gave a single precipitin line with concentrated crude toxin, and it also gave a single arc when tested in immunoelectrophoresis with concentrated crude toxin. A type-specific neutralization effect in monkeys was obtained. Thus, enterotoxin D has been identified with a purified preparation.     

Purification and properties of NADP-dependent isocitrate dehydrogenase from the bovine adrenal cortex

NADP-dependent isocitrate dehydrogenase (EC 1.1.42) was isolated and 430 times purified from the hyaloplasm fraction of bull adrenal cortex using fractionation by ammonium sulphate and acetone, heat treatment, chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-200 and affinity chromatography on 2',5'-ADP-sepharose 4B. The specific activity of homogeneous enzyme is 60 units per 1 mg of protein at 30 degrees C, yield--34%, pH optimum--8.0, molecular weight, determined by gel filtration on Sephadex G-200, is 96 kDa. The preparation electrophoresis in PAAG in the presence of DS-Na reveals one protein fraction with the mobility corresponding to that of protein having molecular weight of 46 kDa. The data obtained evidence for a dimer structure of the isocitrate dehydrogenase molecule from bull adrenals.     

Production of monospecific serum against secretory IgA (anti-SC) and a standard for quantitative analysis]

The authors describe a method of obtaining monospecific serum against secretory IgA and the corresponding standard. An immunochemically pure (11.6S) secretory human IgA was extracted from the colostrum by salt fractionation and gel-filtration through Sephadex G-200 and Sepharose 6B; this IgA was used as an antigen for the immunization and the standard for the quantitative determination of SIgA in the secretions. Monospecific anti-SC-serum was obtained by successive exhaustion of the antiserum against the S IgA immunosorbents prepared from normal human serum and the serum of a patient suffering from A myeloma containing polymeric IgA forms.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Problems associated with the assay of arylsulphatases A and B of rat tissues

A method for the separation and purification of rat liver arylsulphatases A and B by gel filtration on Sephadex G-200 is described. The properties of the A enzyme and its molecular weight are similar to those of the corresponding ox liver enzyme. The B enzymes were found to be dissimilar. The method already developed for the assay of the corresponding enzymes from human tissues was shown to be unsuitable for the assay of the enzymes of rat tissues. A method of assay was developed which permits an approximate determination of the individual rat liver enzymes in a mixture of the two, but precise determination requires prior separation of the enzymes by gel-filtration chromatography.     

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Respiratory behaviour of sea-urchin spermatozoa. II. Sperm-activating substance obtained from jelly coat of sea-urchin eggs.

A sperm-activating substance (SAS) was obtained from the jelly coat of sea-urchin ova and its chemical properties were investigated in three sea-urchin species. The SAS was partially purified from the jelly coat of Pseudocentrotus eggs through several steps of purification by procedures consisting of charcoal adsorption, ion-exchange chromatography on DEAE-Sephadex A-25 column, and gel-filtration on Sephadex G-15 columns. The partially purified SAS was found to contain a ninhydrin-positive material and is inactivated by pronase digestion. The molecular weight of SAS was estimated as about 630 by gel-filtration through Sephadex G-25 and the isoelectric-point of SAS is located at about pH 5.3 by isoelectrofocusing method. The SAS is non-volatile, alcohol-soluble, and labile in a diluted alkaline or acid solution. The origin of SAS is discussed.     

Characteristics of thiamine-binding protein from rat brain synaptosomes

The thiamine-binding protein was obtained from rat brain synaptosomes by affinity chromatography and gel-filtration on Sephadex G-200. The protein is homogeneous by the data of SDS gel-electrophoresis, anode electrophoresis and isofocusing between pH 3.5-9.0. The isoelectric point of this protein is near pH 4.8-5.0. The binding nature of the protein with [14C] thiamine was studied. It is shown that metal ions, especially Na+ and Ca2+, increase the thiamine-binding activity. The binding process is of a saturation character at the thiamine concentrations of 10(-7)-10(-5) M. Thiamine possesses two binding sites with KD1 = 3.1 microM and KD2 = 30 microM. Out of the tested thiamine analogues and antagonists of vitamin B1 thiamine-monophosphate and pyrithiamine were the most competitive.     

Fibroblast mitogens in swine milk include an epidermal growth factor-related peptide

Mammary secretions obtained from lactating sows can support in vitro growth of mammalian cells when added to Dulbecco's Modified Eagle Medium. In order to identify the growth factors present within, sow milk was fractionated and fractions having mitogenic activity were identified by their ability to stimulate DNA synthesis in density-arrested, quiescent (murine) AKR-2B fibroblasts. A prominent mitogenic activity (peak III) was observed in the 3,000-5,000 Mr range. This activity was partially purified by (1) preparative Sephadex G-200 chromatography, (2) gel-filtration in Sephadex G-50 columns and (3) DEAE-cellulose anion exchange chromatography. The last step resolved peak III activity into at least two distinct components. One component was highly-purified by use of reverse-phase high-performance liquid chromatography (RP-HPLC). This activity was identified as an epidermal growth factor (EGF)-related peptide based on its inactivation by polyclonal antibody (IgG fraction) specific for murine EGF (mEGF) and proteolytic agents. The other component is unrelated to EGF. Using cloned mEGF cDNA as a probe, the presence of EGF-related mRNA in lactating mammary tissues and newborn pig small intestine was also demonstrated. These factors may contribute to the preferential growth of gastrointestinal tissues in neonatal pigs.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Properties of delta5-3beta-hydroxysteroid oxidoreductase isolated from Streptomyces griseocarneus.

Delta5-3beta-hydroxysteroid oxidoreductase was extracted in magnesium-containing Tris buffer from sonicated Streptomyces griseocarneus cells. The enzyme was partially purified (150 X) by ion exchange chromatography and gel filtration following (NH4)2SO4 fractionation. Upon gel filtration on Sephadex G-75 to G-200, the greatest part of the activity gave a peak in the fractionation range. The enzyme obtained from the gel yielded small enzyme molecules on repeated chromatography. A molecular weight of 32 to 36 000 was calculated for the activity appearing in the fractionation range of Sephadex G-75 to G-200. The enzyme is highly specific for the irreversible oxidation of the 3beta-hydroxyl group in steroids with a trans-anellated A : B ring system with either C5 or C6 double bond. Delta5-3-ketosteroids are converted into delta5-3-ketosteroids at a high rate, but the isomerase activity cannot be separated from the oxidoreductase activity either by chromatography or by selective heat inactivation. NAD, NADP, FMN or FAD did not influence the activity, but the enzyme is inactive in the absence of molecular oxygen.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

Isolation and purification of NADP-dependent "L-malate"-enzyme from corn leaves]

Isolation and purification of "malic-enzyme" NADP was done using fractionation by ammonium sulfate, anion-exchange chromatography on DEAE cellulose, gel-filtration through Sephadex G-200 and purification on DEAE Sephadex A-50. The isoenzyme isolated had a specific activity of 40-50 mkM/mg protein per min (approximately 80-fold purification) and contained negligible admixtures.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A]

A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Purification and properties of plasminogen activator from human blood plasma after sudden death]

Plasminogen activator from human blood plasma after sudden death was isolated and purified 60-90-fold by precipitation with ammonium sulfate, ZnSO4 and ethanol as well as by chromatography on DEAE-Sephadex A-50 and gel--filtration through Sephadex G-200. The resulting enzyme had specific activity of 110-210 units per mg of protein. The enzyme prepartion possessed no plasmin activity; total content of carbohydrates was 2.4-2.5%; that of syalic acids--1.2-1.3%. The enzyme was found heterogeneous during disc electrophoresis in 7.0% polyacrylamide gel and corresponded in its mobility to beta-globulins of blood plasma. Molecular weight of enzyme as determined by gel-filtration through Sephadex G-200 is 70000. The isoelectric point lies at pH 6.2.