Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.     

Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.     

Purification and characterization of an isoform of protein kinase C from bovine neutrophils

Protein kinase C (PKC) from bovine neutrophils was purified 1420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. The purification procedure from cytosol involved sequential chromatographic steps on DE-52 cellulose, Mono Q, and phenyl-Sepharose. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By filtration on Sephadex G-150, a molecular weight of 85,000 was calculated, indicating that bovine neutrophil PKC in solution is monomeric. Its isoelectric point was 5.9 +/- 0.1. Bovine neutrophil PKC was autophosphorylated in the presence of [gamma-32P]ATP, provided that the medium was supplemented with Mg2+, Ca2+, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of Mr 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of Mr 23,000, which was abundant in the cytosolic fraction of the homogenate, was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of [gamma-32P]ATP, Mg2+, Ca2+, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Casein Kinase-Like Kinase Phosphorylates β-Tubulin and May Be a Microtubule-Associated Protein

The hypothesis that casein kinase II (CKII) is a microtubule-associated protein kinase was investigated using a neuronal cell line and bovine brain. Heparin, an inhibitor of CKII, inhibited the phosphorylation of a PC12 cytosolic protein whose molecular weight was similar to that of beta-tubulin. Partially purified PC12 CKII was immunoreactive to an antibody directed against bovine CKII and was able to phosphorylate purified beta-tubulin in a heparin-inhibitable manner when the concentration of tubulin was less than 50 micrograms/ml. To better determine if CKII is a microtubule-associated protein kinase, bovine brain tubulin was chromatographed on FPLC Mono Q and phosphocellulose columns. Several tubulin casein kinase (TCK) activities were apparent. All TCK activities phosphorylated tubulin and casein, but none was able to phosphorylate the CKII-specific synthetic peptide RRREEETEEE. One of these TCK fractions was immunoreactive to the antibody directed against CKII, and this antibody labeled a 50-kDa molecular mass band that had a molecular mass distinctly different from those of the subunits of CKII. Thus, we suggest that a CKII-like protein, but not CKII, might be a microtubule-associated protein.     

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.     

The O2.- generating oxidase activation of bovine neutrophils. Evidence for synergism of multiple cytosolic factors in a cell-free system

Activation of the O2.- generating oxidase in neutrophils can be achieved with a cell-free oxidase-activating system, which consists of a high speed supernatant (cytosol), a particulate fraction enriched in plasma membrane, GTP-gamma-S, arachidonic acid and Mg ions. Cytosolic proteins from bovine neutrophils were fractionated by chromatography on Mono Q and Mono S columns into two fractions, neither of which was able to activate efficiently the membrane-bound oxidase. However, when combined and added to the cell-free system under optimized conditions, they acted synergistically, activating the oxidase to virtually the same extent as crude cytosol. This synergism demonstrates that more than one cytosolic factor is required for oxidase activation, and can be used to trace the cytosolic factors during the course of their purification.     

Multiple small molecular weight GTP-binding proteins in bovine brain cytosol. Purification and characterization of a 24KDa protein

We have separated multiple small Mr GTP-binding proteins (G-proteins) from bovine brain crude membranes, purified a novel 24KDa G protein (smg p25A) to near homogeneity and characterized it. In this paper, we have studied these small Mr G proteins in the cytosol fraction of bovine brain. [35S]GTP gamma S-binding activity is detected in the cytosol fraction but this activity is one-sixth to one-eighth of that of the crude membrane fraction. When G proteins in the cytosol fraction are purified by successive chromatographies on DEAE-cellulose, Ultrogel AcA-44, hydroxyapatite and Mono Q HR5/5 columns, multiple small Mr G proteins are separated. One of these G proteins shows a Mr of about 24KDa. Its physical, immunological and kinetic properties are indistinguishable from smg p25A. These results indicate that there are also multiple small Mr G proteins in the cytosol fraction of bovine brain, and suggest that one of the cytosol G proteins is the soluble form of smg p25A.     

Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.     

Phosphotyrosyl-protein phosphatases. I. Separation of multiple forms from bovine brain and purification of the major form to near homogeneity

Seven Tyr-protein phosphatase activities were isolated from bovine brain using phosphotyrosyl-casein as a model substrate. The activities were resolved from the cytosolic fraction by a three-step procedure employing successive DEAE-cellulose, phosphocellulose, and gel permeation chromatography steps. The seven activities accounted for 70% of the Tyr-protein phosphatase activity in bovine brain extracts and were distinct from type 1 and type 2 Ser/Thr-protein phosphatases and from the major alkaline phosphatase activities. Apparent molecular weights of the activities by gel permeation chromatography were: phosphotyrosyl-protein phosphatase (PTP)-1A (Mr 86,000), PTP-1B (Mr 24,000), PTP-2 (Mr 88,000), PTP-3 (Mr 90,000), PTP-4 (Mr 80,000), PTP-5 (Mr 48,000), and PTP-6 (Mr 104,000). PTP-5 was the major activity accounting for 26% of total while the remaining activity was divided rather evenly among the other six activities. PTP-5 was further purified to near homogeneity by additional chromatographies on Affi-Gel Blue, heparin-agarose, and Mono S giving an overall purification of 50,000-fold and a yield of 5.8%. One of two major polypeptides (Mr 46,000) in the preparation was identified as PTP-5 since it alone expressed protein phosphatase activity when protein-staining bands were eluted from sodium dodecyl sulfate-polyacrylamide gels and renatured. PTP-5 had a neutral pH optimum, and using phosphotyrosyl-casein as substrate it had a Km of 130 nM and a Vmax of 10 mumol Pi released.min-1.mg protein-1. These kinetic parameters are well within the range of values obtained for other pure protein phosphatases. PTP-5 also dephosphorylated pp60v-src (autophosphorylated at Tyr-416) at 10% of the rate observed with phosphotyrosyl-casein. Additionally the ratio of phosphotyrosyl-casein/pp60v-src phosphatase activity was relatively constant throughout the PTP-5 purification procedure. These results indicate that PTP-5 is able to bind and efficiently dephosphorylate phosphotyrosyl-proteins and suggest that it is a physiologically relevant Tyr-protein phosphatase.     

New purification method for glucocorticoid receptors

In this report, we describe a new purification method for activated recombinant glucocorticoid receptor (GR) utilizing a cation-exchanger (Mono S) at pH8.4. This method is based upon a new finding that activated GR binds to both Mono Q and Mono S columns at the same pH. This method enables us to purify recombinant GR within 3 h. The purified GR represents more than 97% of the eluted proteins. Purified recombinant GR is able to bind specifically to a DNA fragment containing the glucocorticoid response element. Recombinant GR has no tag sequence that can be utilized for purification. Thus, this separation method is also applicable to purification of native GR.     

Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase

Protoporphyrinogen oxidase, the penultimate enzyme in the haem biosynthetic pathway has been purified to apparent homogeneity from bovine liver mitochondria, by a published method (Dailey, H.A. and Fleming, J.E., (1983)), with an additional ion-exchange chromatography step, using a Mono Q column on an FPLC-system. This gave a product with a 68% yield and 870-fold purification. Protoporphyrinogen oxidase (EC 1.3.3.4) has an apparent Mr of 57,000 and the Km for protoporphyrinogen IX was 16.6 microM. Activity of the isolated enzyme was increased by 66% in the presence of oleic acid, and evidence was obtained for a FAD prosthetic group. Ferrochelatase (EC 4.99.1.1) was purified and antibodies were raised in rabbits against ferrochelatase and protoporphyrinogen oxidase, respectively. Anti-protoporphyrinogen oxidase IgG showed marked cross-reactivity with ferrochelatase and anti-ferrochelatase IgG cross-reacted with protoporphyrinogen oxidase. In addition, radiolabelled peptides of both enzymes, generated by chymotrypsin, demonstrated common peptides when analysed by two-dimensional chromatography.     

Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma

Mammalian plasma contains a phospholipase D, which is specific for the glycosyl-phosphatidylinositol anchor found on many eukaryotic cell surface proteins (Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984; Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem. Biophys. Res. Commun. 150, 476-482). We have purified this phospholipase D to homogeneity by a four-step procedure involving a Mono Q and phenyl-5PW columns, followed by wheat germ lectin affinity chromatography and finally another Mono Q column. A 4,500-fold purification was achieved with a 5% yield. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous enzyme has a Mr of 110,000 and appears to consist of a single polypeptide chain. It exhibits identical substrate specificity as compared with the crude preparation, is active over a broad pH range (4.0-8.5), inhibited by the thiol-blocking agent p-chloromercuriphenylsulfonic acid and by 1,10-phenanthroline, and is partially heat-labile.     

Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

The O2(.-)-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP gamma S, and arachidonic acid [Ligeti, E., Doussiere, J., & Vignais, P.V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl[14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5'-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the alpha-subunits of which possess molecular mass values of about 40 kDa.     

Isolation of three types of Gi from bovine spleen

We have previously reported the purification of two alpha subunits of G proteins, Gi2 and Gi3, from bovine spleen. However, it recently became clear that the preparation of Gi3 alpha contained a significant amount of Gi1 alpha by the immunoblot analysis using specific antibodies. In this study, we purified these G proteins as a trimer form from bovine spleen, and obtained following results. (1) Gi3 was separated from Gi1 using Mono Q column chromatography. Isoelectric focusing was employed to distinguish Gi3 from Gi1 in the column eluates. (2) Purified Gi2 and Gi3 retained much higher activities to bind GTP gamma S or to be ADP-ribosylated by pertussis toxin than the alpha subunits purified previously. (3) Using these spleen Gi2 and Gi3 and bovine brain Gi1, the parameter of GTP gamma S binding to the three types of Gi was compared. Three Gis showed different rates of GTP gamma S binding but showed the similar Kd values.     

Purification of Dictyostelium myosin II heavy chain kinase A based on the increase in negative charge accompanying hyperphosphorylation

The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).     

Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), which catalyzes the C-20 methylation of precorrin-2 to precorrin-3, was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain. Ammonium sulfate fractionation followed by chromatography on DEAE-Trisacryl, hydroxyapatite, and Mono Q HR purified the enzyme about 110-fold, with a 28% yield. For enzyme purification and characterization, a coupled-enzyme assay was developed which generated in situ the highly oxygen-sensitive substrate, precorrin-2, from delta-aminolevulinic acid. Evidence is given that the chemically reduced form of sirohydrochlorin (dihydrosirohydrochlorin) is methylated at C-20 to precorrin-3 by pure SP2MT. No subsequent SP2MT-dependent methylation reaction of precorrin-3 was detected. The native enzyme has an apparent molecular weight of 53,000, as estimated by gel filtration, and consists of two identical subunits of Mr 26,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stepwise Edman degradation provided the N-terminal sequence of the first 17 amino acids.     

Purification of Dictyostelium myosin II heavy chain kinase a based on the increase in negative charge accompanying hyperphosphorylation

The initial step in the purification of Dictyostelium myosin 11 heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg²⁺ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 m KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M_r greater than 700,000).     

Protein phosphatase type 1 catalytic subunit forms nondissociable dimers

Protein phosphatase type 1 is the major enzyme in skeletal muscle and liver for the dephosphorylation of Ser(P) and Thr(P) phosphoproteins. The cDNA for the catalytic subunit encodes a polypeptide of Mr 35,400 kDa, consistent with the Mr of 36,000-38,000 of the active protein purified in various laboratories. However, several investigators have found a Mr 70,000 protein for phosphatase type 1. In this report proteins of Mr 38,000 and 70,000 were resolved by Mono Q chromatography after extensive copurification from rabbit skeletal muscle. Antibodies affinity-purified against a type 1 phosphatase catalytic fragment reacted with both proteins in Western immunoblotting. Fractions from each peak were cleaved with cyanogen bromide and the major peptides were the same size by electrophoresis in gradient polyacrylamide gels. Cyanogen bromide peptides of the individual bands also were mapped by reversed-phase high-performance liquid chromatography. The purified Mr 38,000 and 70,000 proteins had identical HPLC peptide maps and also gave the same amino acid compositions after acid hydrolysis. Purified Mr 38,000 phosphatase catalytic subunit spontaneously formed a Mr 70,000 dimer that resisted usual dissociation conditions, i.e., boiling dodecyl sulfate plus 2-mercaptoethanol, but could be cleaved to about half size by various proteases, indicating that monomers were bound together near their amino or carboxy termini. Physiological changes in protein phosphatase type 1 are reflected in the amount of nondissociable dimers detected in tissue extracts.     

Purification of an Acid Invertase from Washed Discs of Storage Roots of Red Beet (Beta vulgaris L)

The purification of an acid invertase from washed discs of storageroots of red beet (Beta vulgaris L.) is described. An overallpurification of 1210-fold was obtained using a combination of(NH₄)₂SO₄ precipitation, size-exclusion chromatography, ion-exchangechromatography, conA-sepharose chromatography and two roundsof FPLC on Mono Q HR 5/5, the first at pH 7·5, the secondat pH 6·5. The purified enzyme had a specific activityof 206