An apparent oligomer of malate dehydrogenase from bean leaves

Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm³.     

Dopaminergic agonist and antagonist effects on striatal tyrosine hydroxylase distribution

Gamma-aminobutyric acid (GABA) and benzodiazepine binding sites solubilized from rat cerebral cortex were not separated by ammonium sulfate fractionation, gel filtration, sucrose density gradient centrifugation and DEAE-cellulose chromatography. The molecular weight of both binding sites was determined to be 670,000 by gel filtration and the sedimentation coefficients to be 11.3S by sucrose gradient centrifugation. Scatchard plots of the binding of GABA, muscimol and flunitrazepam with Sepharose 6B eluate indicated that their receptors had a single class of sites for each ligand, and the maximum number of binding sites for GABA and muscimol was all but equal and double of that for flunitrazepam. Flunitrazepam binding was enhanced by GABA agonists.     

家兔肝脏LRP蛋白的分离纯化与鉴定

利用家兔LRP蛋白聚集形成蛋白复合物的性质,首先通过两次蔗糖密度梯度离心从家兔肝脏中分离得到LRP粗提液,再利用分子大小差异,使用Sephacryl-1000分子筛层析纯化,SDS-PAGE银染结果显示最终得到了单一电泳条带的家兔LRP蛋白.最后使用LRP单克隆抗体的Western blotting鉴定结果正确.     

Phosphatidylcholine-promoted interaction of the catalytic and regulatory proteins of adenylate cyclase

The catalytic protein of rabbit hepatic adenylate cyclase, after chromatographic separation from the GTP-binding regulatory protein (G/F) (Ross, E. M. (1981) J. Biol. Chem. 256, 1949-1953), is essentially free of endogenous phospholipids. This preparation is active in the presence of Mn2+ and is markedly stimulated by forskolin, but it is stimulated only slightly by the addition of purified G/F plus an activator (GTP gamma S or NaF). The ability of activator-liganded G/F to stimulate the activity of the resolved catalyst is increased up to 8-fold by the addition of either dimyristoylphosphatidylcholine (0.3-1.5 mM optimal concentration) or several other phosphatidylcholines. Phosphatidylcholine stabilizes the catalyst to denaturation but has little effect on its basal activity or on its stimulation by Mn2+ or forskolin. It also had no stimulating effect on the activation of G/F by GTP gamma S. These data are interpreted as showing that phosphatidylcholine promotes or is required for the stimulatory interaction of activator-liganded G/F with the catalytic protein of adenylate cyclase. Lubrol 12A9, Triton X-100, cholate, lysophosphatidylcholine, digitonin, and phosphatidylserine could not substitute for phosphatidylcholine. The detergents inhibited stimulation by liganded G/F even in the presence of phosphatidylcholine. Removal of cholate from a mixture of soluble catalytic protein and phosphatidylcholine by dialysis and sucrose density gradient centrifugation caused the binding of catalytic protein to large unilamellar vesicles. This preparation was further reconstituted with increasing amounts of G/F to yield vesicles with varied G/F:catalyst ratios and similarly varied responses to G/F-mediated activating ligands.     

Isolation of latent phenoloxidase from prepupae of the housefly,Musca domestica

Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.     

Resolution of microsomal membranes into fractions differing in polypeptide composition

Microsomes from rat liver, prepared by gel filtration, were subjected to centrifugation in a continuous sucrose density gradient containing a low concentration of deoxycholate. The membranes were subfractionated into five bands differing in appearance and equilibrium density. Each band, when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, displayed a characteristic population of membrane proteins.     

Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells. Subunit structure and molecular weight determination.

Cytochrome c oxidase was purified from mitochondria of Candida utilis yeast cells. The purification procedure involved the hypotonic incubation of mitochondria followed by washes with increasing concentrations of KCl. The membrane fragments derived from this procedure were subjected to ammonium sulfate fractionation in the presence of 2% cholate. The purified active enzyme contained 8.5–9.2 nmol heme a per mg protein and was free of other types of hemoproteins. Upon Sephadex G200 gel filtration in the presence of cholate, an apparent molecular weight of 200,000 was estimated. A single band was observed for the active enzyme upon DEAE-cellulose chromatography, sucrose density gradient centrifugation, and Sephadex G200 gel filtration.Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six polypeptide bands with apparent molecular weights of 49,000, 32,000, 28,000, 20,000, 13,500, and 8,000, respectively. The six components were also resolved by gel filtration on Sephadex G200, equilibrated with 0.1% sodium dodecyl sulfate, giving apparent molecular weights of 46,000, 35,000, 23,000, 19,000, 12,500, and 7,800.     

Purification and characterization of chaperonin 10 from Chromatium vinosum.

Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.     

Reassembly in vitro of lung surfactant lipoprotein

This report describes a successful attempt to reassemble, $\text{in vitro}$, two fractions obtained from bovine lung surfactant lipoprotein. An apoprotein isolated by gel filtration in the presence of sodium deoxycholate was recombined with lipid extracts of the surfactant, in a highly alkaline buffer (pH 10) containing 10 mM sodium deoxycholate. Sonication, dilution 1 to 10, dialysis, and washing by means of centrifugation were used to produce a lipid-protein complex. Centrifugation in a continuous sucrose density gradient revealed that this material had a density of 1.081 gm/ml and a phospholipid/protein ratio respectively almost the same as those of the original lipoprotein.     

Characterization of the molecular form of cardiac phospholamban

The native form of phospholamban is not known and it is presently under debate whether this protein exists as a monomer or an oligomer in cardiac sarcoplasmic reticulum. The currently accepted model for phospholamban is pentameric, based primarily on its behavior in SDS-polyacrylamide gel electrophoresis. In this study, sucrose density gradient centrifugation and gel filtration chromatography were used to determine the form of phospholamban under nondenaturing conditions. Purified phospholamban or phospholamban present in solubilized cardiac sarcoplasmic reticulum was centrifuged through 5–20% sucrose density gradients in the absence or presence ofn-octylgucoside. The sucrose density gradient fractions were assayed for acid precipitable³²P-incorporation in the presence of [-³²P]ATP and cAMP-dependent protein kinase catalytic subunit.³²P-containing peak fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis, using a phospholamban-polyclonal antibody, to confirm the presence of phospholamban. Purified phosphoblamban migrated with an apparent molecular weight of 25,000 daltons in the sucrose gradients in either the absence or presence of detergent. Phospholamban present in solubilized cardiac sarcoplasmic reticulum migrated with a similar apparent molecular weight when detergent was included in the sucrose gradients. In addition, solubilized cardiac sarcoplasmic reticulum was subjected to gel filtration chromatography in the presence of deoxycholate. Under these conditions phospholamban migrated with an apparent molecular weight of 24,500 daltons. These data suggest that phospholamban prefers an oligomeric assembly and this may be the form present in cardiac sarcoplasmic reticulum membranes.     

易发生聚集的重组HBcAg病毒样颗粒的纯化*

目的:探索针对易发生聚集的重组HBcAg病毒样颗粒(VLP)的有效纯化方案。方法:培养的大肠杆菌经IPTG诱导重组HBcAg蛋白的表达,菌体超声破碎后的离心沉淀用含有不同浓度尿素的PBS缓冲液重悬溶解,经密度梯度离心并结合电镜观察对VLPs行为进行分析鉴定。以Sepharose 4 FF凝胶过滤层析在选定的尿素条件下纯化沉淀溶解液,纯化获得的目的蛋白进一步在含30%山梨醇的PBS中脱盐去除尿素。整个过程以SDS-PAGE及电镜进行各步骤样品中目的蛋白的分析。结果:含有1mol/L尿素的PBS缓冲溶液重悬超声沉淀,可有效溶解聚集的VLPs,在蔗糖密度梯度离心中显示典型HBcAg VLPs的行为,且电镜观察颗粒形态结构完整。经1mol/L尿素下凝胶过滤,VLPs进一步获得纯化。在脱尿素过程中流动相采用含30%山梨醇的PBS,有效避免了VLPs在尿素去除后重新聚集。结论:尿素与山梨醇的联合应用,为具有聚集现象的VLPs纯化制备提供了一种有效解决方案。     

Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins.

Phosphatidylinositol (PtdIns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 micrograms/ml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycan-specific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Association of three chicken proteins with the 34 kD target of rous sarcoma virus tyrosine kinase

Transformation of chicken embryo fibroblasts by avian retroviruses induces the tyrosine phosphorylation of a 34-39 kD cellular protein (p34). In vitro, p34 isolated from intestine interacts with F-actin in a Ca2+-dependent manner. We report here that, in the absence of Ca2+ chelators, three proteins co-purified with p34 extracted from a cytosolic or membrane fraction of chicken embryo fibroblasts; these two fractions account respectively for 10-20% and 50% of the total cellular p34. Isolated from the cytosoluble fraction of fibroblasts by sucrose gradient centrifugation and hydrophobic chromatography, p34 and the other proteins behaved as a homogeneous species upon non-denaturing gel electrophoresis, gel filtration, and CsCl density gradient centrifugation, thus indicating a strong association. Moreover, an analysis by electron microscopy following uranyl acetate staining revealed particles with a raspberry-like shape. This association was always disrupted by the calcium-chelating agent, EGTA.     

Inhibitor-1 phosphatase activity in vascular smooth muscle

The histone-H1 and polylysine stimulated "latent" phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with beta-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.     

Isolation of a prokaryotic photoreceptor: sensory rhodopsin from halobacteria

The photoreceptor sensory rhodopsin was isolated from halobacterial cell membranes solubilized in laurylmaltoside. In the presence of retinal, detergent and salt the native protein was obtained in pure form by sucrose density gradient centrifugation, hydroxyapatite chromatography and gel filtration. The apparent mol. wt of the molecule was 24 kd if analyzed by SDS gel electrophoresis, and 49 kd by sedimentation and size-exclusion chromatographic analysis. The chromoprotein had an absorption maximum at 580 nm which was 8 nm blue-shifted compared to the membrane-bound state. The molecule was photochemically active and the action spectrum for formation of SR380, the long-lived intermediate, coincided with the absorption spectrum.     

大麦根质膜微囊的制备及其H~ ,Ca~(2 )转运活性

用蔗糖密度梯度离心法制备出密闭程度较高的大麦根细胞质膜微囊。喹吖咽荧光猝灭和~(45)Ca~(2 )同位素示踪测定表明所制备的微囊具H~ ,Ca~(2 )转运活性。对制备出的质膜制剂纯度和膜朝向进行了分析,并探讨了质膜纯化中影响膜微囊密闭性的因素。匀浆液和悬浮液巾的单价离子盐有利于密闭膜微囊的形成。蔗糖密度梯度和葡聚糖密度睇度离心法均可得到密闭性较高的膜微囊,但后者的纯化效果较差。     

Purification and characterization of porcine heart type 2A protein phosphatases.

Protein phosphatases 2A1 and 2A2 were isolated from porcine heart tissue extracts by precipitation at pH 5.0 and separated by chromatography on DEAE-Sephacel. Phosphatase 2A1 was then purified to apparent homogeneity by chromatography on phenyl-Sepharose, aminohexyl-Sepharose, Sephacryl S-300, and L-tyrosine-agarose. Phosphatase 2A2 was purified to apparent homogeneity by chromatography on phenyl-Sepharose, DEAE-Sephacel, aminohexyl-Sepharose and L-tyrosine-agarose. Purified phosphatases 2A1 and 2A2 had specific activities of 2200 and 2710 nanomoles of phosphate released from phosphorylase a/mg protein, respectively. The apparent molecular weights of phosphatases 2A1 and 2A2 on gel filtration were 155 and 105 kDa, respectively. Both enzymes contain 70 and 37 kDa subunits and 2A1 also contains a 57 kDa subunit. The 37 kDa catalytic subunit (2Ac) was obtained from the purified phosphatases by treatment with room temperature ethanol followed by sucrose density gradient centrifugation or gel filtration chromatography.     

Concanavalin A is not a ferritin

Contrary to recent claims, in vitro evidence has been obtained to establish that Concanavalin A (Con A) is not a ferritin. Four techniques including immunoprecipitation, gel filtration, sucrose density gradient ultracentrifugation and CsCl centrifugation were employed. None of them showed that Con A is a ferritin.     

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.