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.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.     

Purification of Oidiodendron kalrai proteases.

Oidiodendron kalrai yeast-phase cells demonstrate proteolytic activity. Some of the proteolytic enzymes of the crude extract were purified by a combination of ammonium sulfate precipitation, Sephadex G-200, and diethylaminoethyl (DEAE) cellulose column chromatography. At least six proteins exhibiting a range of proteolytic activities could be identified by these procedures. Purity of the enzyme fractions obtained from the DEAE-cellulose columns was tested by running polyacrylamide gels.     

Characterization of multiple forms of histone phosphatase in rat liver.

By using chromatography on DEAE-cellulose, aminohexyl-Sepharose 4B and Sephadex G-200, rat liver extract was shown to contain at least three fractions, IA, IB and II, of histone phosphatase. Fractions IA and II are probably the same enzymes as the previously described glycogen synthase phosphatase and phosphorylase phosphatase, respectively, but IB exhibits noticeable activities only with phosphohistone as substrate. Approximate molecular weights of 69 000, 300 000 and 160 000 were determined by gel filtration on Sephadex G-200 for IA, IB and II, respectively.     

Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.     

Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with (14)C-labelled carbohydrate or with (3)H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-(14)C]glucose or [G-(3)H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s(0) (25,w) values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B-C, which were chemically, biosynthetically and immunologically very similar.     

Regulatory effects of S-100 protein and parvalbumin on protein kinases and phosphoprotein phosphatases from brain and skeletal muscle

Summary In the eluted fractions of histone-treated crude extracts separated by Sephadex G-200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio-Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine.     

Non-specific activation of mouse peritoneal macrophages by a freshwater ciliate, Tetrahymena pyriformis

Toxoplasma-killing activities of mouse peritoneal macrophages activated by the extracts of Tetrahymena pyriformis (Korean and Chinese strains) were evaluated, and the active protein fractions from both strains were partially characterized by a method including chromatographies and SDS-PAGE. The first peak in Korean strain and the second peak in Chinese strain of T. pyriformis obtained by DEAE-Sephadex A-50 chromatography were most effective in the activation of macrophages to kill Toxoplasma gondii tachyzoites in vitro. Subsequent fractionations of obtained peak fractions were performed on a Sephadex G-200 gel. The first peaks fractionated from both strains of T. pyriformis had the highest toxoplasmacidal activities, and when subjected to the SDS-PAGE, one prominent band was visualized for each of the strains showing the same molecular weight of ca. 52.6 kDa. This active protein is suggested to be related to non-specific activation of mouse peritoneal macrophages.     

Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.

Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.     

Isoenzymes of Starch Phosphorylase in Triticale

Starch phosphorylase (α-1,4 glucan: orthophosphate α-D-glucosyl transferase, EC 2.4.1.1) was partially purified from triticale by ammonium sulphate fractionation and chromatography on Sephadex G-200. Three active fractions were obtained, which differed in their kinetic properties and activities in the presence of certain glycolytic and Krebs cycle intermediates. These fractions may have specific functions in starch synthesis during grain development. The isoenzymic fraction Ell, in particular, may have a regulatory function by controlling formation of primers from oligosaccharides.     

Myrosinase in Brassicaceae: I. LOCALIZATION OF MYROSINASE IN CELL FRACTIONS OF ROOTS OF Sinapis alba L.

Myrosinases (thioglucoside glucohydrolases E.C. 3.2.3.1 [EC] .), whichcatalyse the hydrolysis of glucosinolates present in Brassicaceae,were isolated from Sinapis alba L. seeds. The crude enzyme extractwas purified using gel and ion-exchange chromatography, isoelectricfocusing, and polyacrylamide gel electrophoresis. The separationof two myrosinase isoenzymes was obtained after gel chromatographyon Sephadex G-100. Further purification of the main myrosinasecomponents was achieved when the combined isoenzymes were separatedon the anion-exchanger DEAE-Sephadex A-50 followed by polyacrylamidegel electrophoresis. A similar purification was obtained when the crude extract wasgroup-fractionated on Sephadex G-50 followed by DEAE-cellulosechromatography on Whatman DE-52 and gel chromatography on SephadexG-200. The enzyme from the last step was further separated byisolectric focusing into two isoenzymes with isoelectric points4.9 and 6.2. In order to clarify where the myrosinase was localized in theroot tip cells, cell fractionation studies were performed usingaldehydes as pre-fixatives to stabilize the enzymes and thecell organelles. Biochemical tests of crude and purified samplesof the isolated myrosinases showed that when glutaraldehydeor formaldehyde were used as pre-fixatives at a final concentrationof 1% (w/v), they did not inhibit the enzyme activity. Relativelyhomogeneous cell organelle fractions were obtained using ultracentrifugationand stepwise sucrose gradients. The myrosinase activity expressedon the basis of the protein content was found to be highestin the dictyosome and smooth endoplasmic reticulum fractions     

Simple radioassay for uridine phosphorylase and 5'-nucleotidase

A method is described whereby a large number of chromatographic fractions containing protein or enzyme components may be run on a single-starch gel. A multiple punch for making the sample holes, gel slab support, and a gel slicer used to cut several slices from the one gel for staining for the various enzyme or protien constituents are described. An example is given of a run using fractions obtained from a Sephadex G-200 column chromatography run of a herring muscle extract. Densitometry may be used to assay the stained gels. In another example a commercially obtained muscle lactate dehydrogenase preparation was chromatographed on a Sephadex G-200 column. The electrophoretic run of the fractions revealed the presence of both heart-type A and muscle-type B lactate dehydrogenase subunits and showed that the column run had effected a partial separation of the various tetramers.     

Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.     

Multiple molecular forms of murine thymocyte-stimulating factor

Murine thymocyte stimulating factor (TSF) was found to sediment in sucrose density gradients on a broad band with peaks at about 2.60 S and 2.0 S. Two main peaks of TSF activity (with buoyant densities of 1.34 and 1.28 g/ml) were found in CsCl density gradients. Gel chromatography on Sephadex G-100 columns of the material sedimented in sucrose or CsCl density gradients originated multiple peaks of TSF activity with various molecular weights. Heterogeneity of molecular forms of TSF was also found upon dilution of the factor. The lowest molecular weights found were 4000 and 4700 daltons. When Sephadex fractions containing the low molecular weight material were pooled and rerun on Sephadex columns, molecular species with a wide range of molecular weights were found. Temperature also affects the appearance of the low molecular weight forms of TSF. Most of the experiments presented in this work were carried out with Sephadex-purified TSF. Multiple molecular forms, however, and, in particular, the forms with molecular weights of 4000 and 4700 daltons were found also with TSF-Fraction IIIa, a highly purified preparation of this factor.     

The synthesis and turnover of rat liver peroxisomes. I. Fractionation of peroxisome proteins

Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.     

Preparation and description of high-molecular-weight soluble surface antigens from a group A Streptococcus

High-molecular-weight proteins having M protein reactivity were isolated without acid or alkaline digestion. Treatment of a heat-killed group A Streptococcus with sonic vibration released antigens which reacted strongly and specifically with absorbed type-specific antiserum. This antigen preparation was released without diminishing the total yield of acid-extractable M protein of the original heat-killed cells. Fractionation of the sonic preparation on a sucrose gradient yielded four peaks of M reactivity. When these fractions were placed on Sephadex G-200 columns, the M reactive material of three fractions appeared in the void volumes, suggesting that the active material in each had a molecular weight greater than 300,000. The reactivity of the fourth fraction followed closely the void volume of Sephadex G-100. Chemical analysis revealed heterogeneity of the fractions. Spectral analysis showed virtual absence of nucleic acid in three of the fractions and a moderate amount in the fourth. Bactericidal inhibition tests showed activity of three of the four fractions. Analysis of the fractions by Ouchterlony double-diffusion technique revealed that each of the four fractions had several antigenic constituents. All four contained M antigen. T antigen and a third unnamed antigen were present in some of the fractions. Group reactivity was present in all fractions, but did not reside on the M molecule. The enhanced potential of sonically released antigens to induce high-titer specific precipitating antibodies to M protein is discussed.     

An antigen common to a wide range of bacteria. I. The isolation of a 'common antigen' from Pseudomonas aeruginosa

In crude water-soluble extracts of Pseudomonas aeruginosa 64 antigens can be demonstrated by crossed immunoelectrophoresis in agarose with polyvalent Pseudomonas-immunoglobulin. One of these antigens cross-reacts with antigens prepared from bacteria of a wide range of taxonomic groups. Monospecific immunoglobulins to this antigen (Common Antigen) were produced by immunization with the appropriate immunocomplex extracted from agarose. Common Antigen was purified by the combination of two fractionation methods: Precipitation of the crude extract with 18% (w/v) sodium sulfate, followed by gel filtration of the supernatant on a Sephadex G-200 column. By this method, 35% of Common Antigen from the crude extract was recovered, more than half of the fractions electrophoretically pure. Electrophoresis of reduced Common Antigen on a dodecyl sodium sulfate polyacrylamide gel revealed two protein bands with apparent molecular weights of 59-62 000 and 62-65 000, respectively. The untreated antigen, however, passed a column of Sephadex G-200 with the void volumen, indicating a substance of high molecular weight (> 4-600 000).     

Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes

A lipid transfer protein was isolated from bovine liver. Following the release of soluble proteins from liver microsomes, the transfer protein was purified 75-fold to near homogeneity by a combination of DEAE-cellulose ion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. About 7% of the original activity was recovered. The purified fraction promoted the transfer of triglyceride, cholesteryl ester, phosphatidylcholine, and phosphatidylethanolamine. When the fractional rates of lipid transfer were compared, the transfer of apolar lipids was over 10 times faster than that of phospholipid. The purified transfer complex contained less than 5% lipid. No carbohydrate was detected. Electrophoresis of the purified protein on polyacrylamide gels under non-denaturing conditions showed a single band. Elution of protein from slices of unstained gels showed that lipid transfer activities coincided with the position of the protein band on the stained gel. When the purified protein was electrophoresed in the presence of SDS, two bands, accounting for more than 95% of the staining density, were observed with molecular weights at 58 000 and 88 000. The purified transfer protein eluted from a Sephadex G-200 column at a position corresponding to a protein with a molecular weight of 220 000, which probably represents a complex of two or more polypeptides. The purified transfer protein was activated by increasing NaCl concentrations up to about 100 mM. At higher NaCl concentrations the transfer activity decreased. Maximal transfer activities were observed at pH 7. The protein was inactivated by heating above 50 degrees C. The transfer rates were not greatly increased by changing the assay temperatures between 20 degrees C and 50 degrees C. These activity characteristics of the transfer protein were the same whether triglyceride or cholesteryl ester transfer activities were measured.     

Purification and characterization of fish surface mucin

Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present.