Glycerol Density Gradient Sedimentation Research Articles

Separation and Partial Characterization of Three Distinct Intracellular GLUT4 Compartments in Rat Adipocytes: SUBCELLULAR FRACTIONATION WITHOUT HOMOGENIZATION

Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were approximately 25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to approximately 20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.

Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA.

Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients. One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P. aeruginosa chromosomal map. The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family. On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy. Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P. aeruginosa strains isolated from cystic fibrosis patients. We expressed and purified the protein products from the mucA and mucB open reading frames. The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo. Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.

Major component of Ra-reactive factor, a complement-activating bactericidal protein, in mouse serum.

A complement-activating bactericidal protein, Ra-reactive factor was isolated from mouse serum by an affinity method. The m.w. of the isolated RaRF estimated by glycerol density gradient sedimentation (around 300,000) was the same as that of the active material in mouse serum. As evidenced by gel filtration, the intact RaRF was decomposed into high (higher than 200,000) and low (50,000 to 200,000) m.w. components by treatment with 10% acetonitrile. SDS- and acid/urea-PAGE demonstrated that the high m.w. component was completely dissociated into equimolar quantities of two kinds of 28 kDa polypeptides, P28a and P28b, under reducing conditions, indicating that the association of these polypeptides was stabilized by disulfide bonds. The ability to bind specifically to the Ra determinant was retained in the high m.w. component, although the complement-activating potency was lost. The amino acid compositions of P28a and P28b polypeptides were compared with those of related serum proteins. The P28a and P28b polypeptides were found to have the highest homology to rat mannan-binding protein and mouse and human C1q subcomponent of complement.

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.

Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung.

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.

Large-scale isolation of the Neurospora plasma membrane H +-ATPase

A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O 2 to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H +-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with α-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H +-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 μmol of P i released/min/mg of protein at 30°C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.

Biochemical and immunological properties of the reverse transcriptase associated with a hamster retrovirus.

Several properties of an RNA-directed DNA polymerase associated with a hamster retrovirus (HaRV) were examined and found to be similar to other polymerases from mammalian type-C viruses in that the enzyme (i) is more active with Mn2+ than Mg2+, (ii) uses the reverse transcriptase-specific poly(rCm).oligo(dG) template, (iii) possesses substantial endogenous polymerase activity and (iv) is strongly inhibited by homologous antisera and moderately inhibited by antisera directed against other type-C viruses. In contrast to previous reports of polymerases from other hamster viruses, HaRV polymerase is active in endogenous assays and the activity is associated with a 70,000 mol. wt. polypeptide in highly purified virions and with 70,000 and 85,000 mol. wt. polypeptides in fresh, unpurified virus. Only one major peak of polymerase activity eluted from DEAE-cellulose while subsequent elution of this peak from phosphocellulose produced two major peaks of polymerase activity. The mol. wt. of these two peaks were 70,000 and 85,000 by glycerol density-gradient sedimentation. The HaRV reverse transcriptase and p30 were found to be most closely related antigenically to other rodent retrovirus proteins.

Calicivirus intracellular RNA: Fractionation of 18–22 S RNA and lack of typical 5′-methylated caps on 36 S and 22 S San Miguel sea lion virus RNAs

Radiolabeled RNA from Vero cells infected with a calicivirus, San Miguel sea lion virus type 2, in the presence of actinomycin D, was fractionated by glycerol density gradient sedimentation to yield 36 S single-stranded RNA (ssRNA) and heterogenous RNA sedimenting in the 18–22 S region. Further fractionation of 18–22 S RNA by LiCl precipitation, CF-11 cellulose chromatography, and polyacrylamide gel electrophoresis yielded 22 S ssRNA, two doule-stranded RNAs )dsRNA), and a partially single-partially double-stranded RNA (ss/dsRNA). Sedimentation analysis indicated the major and minor dsRNAs were equivalent to two strands of 36 S and 22 S ssRNAs, respectively, and the dsRNA core of ss/dsRNA was equivalent to the major dsRNA. Neither 22 S ssRNA (presumed subgenomic mRNA) nor 36 S ssRNA (presummed full genomic RNA plus intracellular virion RNA) had the typical 5′-methylated cap.

RNA synthesized in calicivirus-infected cells is atypical of picornaviruses

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.

Purification and characterization of 3':5'-cyclic GMP-dependent protein kinase.

Guanosine 3':5'-cyclic monophosphate (cGMP)-dependent protein kinase has been purified to homogeneity from bovine lung by affinity chromatography and characterized. Partially purified protein kinase, specifically activated by low concentrations of cGMP (22 NM), was adsorbed onto 8-(2-aminoethyl)-amino-adenosine 3':5'-cyclic monophosphate-Sepharose. After washing to remove nonspecific proteins, cGMP-dependent protein kinase was specifically eluted by 0.1 mM cGMP. The purified protein contained cGMP-dependent protein kinase and specific cGMP binding activities. Purification of the holoenzyme was possible because subunit dissociation does not occur upon cyclic nucleotide binding. cGMP-dependent protein kinase holoenzyme has an apparent molecular weight of 150,000 as determined by glycerol density gradient sedimentation. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band of 71,000 molecular weight was observed that suggested the holoenzyme is a dimer composed of subunits of identical molecular weight. cGMP-dependent protein kinase required high concentrations of Mg+2 for optimal activity; a heat-stable protein kinase modulator which inhibited adenosine 3':5'-cyclic monophosphate-dependent protein kinase activity had no effect on the activity of purified cGMP-dependent protein kinase.

Replication of T4 DNA in vitro. II. Assay system for and some properties of gene products required for T4 DNA replication.

[3H]dTTP was not incorporated into T4 DNA in the in vitro system for T4 DNA replication when the system was prepared from cells infected with T4 amber mutants defective in DNA replication. [3H]dTTP incorporation was resumed by adding the missing gene product to the defective system. DNA replication by the reconstituted system proceeded by the discontinuous mode of replication, as observed in the wild-type system. By using this in vitro complementation system, molecular weights of gene 41, 43, 44, 45, and 62 products in the active form were roughly estimated as 60,000, 130,000, 130,000, 60,000, and 130,000, respectively. Complex formation between the products of genes 44 and 62 was detected. Other strong interactions between the gene products tested were not detected by glycerol density gradient sedimentation. Interaction of gene products with denatured DNA was analyzed by using a DNA-agarose column, and the results showed that products of genes 32 and 43 had a strong affinity for DNA.