Non-dissociating Conditions Research Articles

Purification and properties of a soluble inorganic pyrophosphatase from Rhodopseudomonas palustris.

A soluble inorganic pyrophosphatase from photolithoautotrophically grown Rhodopseudomonas palustris was purified to a state of apparent homogeneity applying high resolving liquid chromatography steps. Values of 65 500 and 64 500 were calculated for the relative molecular mass under non-dissociating conditions employing gel filtration and high-performance liquid chromatography, respectively. Dissociation sodium dodecyl sulfate gel electrophoresis resulted in a value of 32 000, indicating that the enzyme is composed of two subunits of equal molecular mass. Isoelectric focusing revealed a pI value of 4.7. The purified enzyme was specific for PPi and the activity was modified by divalent cations. Ca2+, Mn2+, Mg2+ and Co2+ were potent activators at a concentration ratio of [Me2+]/[PPi] less than 1. Ca2+ turned out to be the most potent activator. Free Me2+ was inhibitory on the PPiase activity. The (Me-PPi) complex is regarded as the functional substrate. Km and Ki values of the metal activation and inhibition were determined. An activation energy of Ea = 14.4 kJ/mol was derived from Arrhenius plots for the enzymatic reaction.

Charge and size of mesangial IgA in IgA nephropathy

To characterize the physicochemical properties of the mesangial IgA in primary IgA nephropathy, acid-eluates from percutaneous renal biopsies of 20 patients were examined. The acid-eluates were obtained from 1287 +/- 498 glomerular sections. The IgA content (mean 15 +/- 10 ng) represented 0.4% of the total eluted proteins. To analyze the molecular weight and the charge of eluted IgA, 11 eluates were subjected to high pressure liquid chromatography (at pH 6.8 and/or pH 3.5) and five eluates to isoelectric focusing on agarose. IgA was detected in the fractions by an IgA-RIA. Comparison of the elution profiles at different pH showed a statistically significant decrease of the excluded IgA peak (greater than or equal to 1,000,000 daltons), and a significant increase of polymeric IgA peaks (1,000,000-320,000 and 320,000 daltons) in acidic chromatography, as compared to non-dissociating conditions. Under acidic conditions, polymeric IgA represent 64% of total eluted IgA. Secretory component binding to polymeric IgA was demonstrated in four out of eight eluates tested. The isoelectric point (pI) of eluted IgA ranged from 4.5 to 5.6, contrasting with the broader and more neutral pI of normal serum IgA (4.5 to 6.8). This study shows that the multimeric nature of IgA, the formation of IgA complexes, and the anionic charge of IgA are likely to be involved in the mesangial IgA deposition in idiopathic IgA nephropathy.

What family of ATPases does the vacuolar H+-ATPase belong to?

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [ 14C]DCCD and [ 14]NEM bound to a 9 kDa polypeptide, while [ 14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [γ- 32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H +-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H +-ATPase of yeast.

Fc intermediate (Fc I), a papain-generated fragment of human IgG, intermediate in charge, molecular weight and cleavage between the Fc and Fc' fragments of IgG

Fc intermediate (Fc i) is a papain-generated fragment of human IgG which is intermediate in charge, mol. wt and state of cleavage between the Fc and Fc' fragments of IgG. It is composed of two polypeptide chains of unequal mol. wt held together by non-covalent bonds between the Cγ3 regions. The larger polypeptide chain has both a Cγ2 and Cγ3 domain and its N-terminus is at Leu 235 (60%) and Leu 234 (40%) (IgG1 Eu numbering). The smaller polypeptide chain is composed of a Cγ3 domain with its N-terminus at Gly 341. The carboxy-termini obtained by carboxypeptidase digestion and by a computer program which determined the most probable sequences by fitting the amino acid compositions to the sequence of IgG Eu Fc were heterogeneous involving residues 440–446 for the large polypeptide chain and 429–436 for the small one. The calculated mol. wt of the large polypeptide chain was 26,183, assuming the N-terminus at Leu 234 and C-terminus at 446 and including the carbohydrate moiety. The calculated mol. wt for the small polypeptide chain was 10,682, with the N-terminus at 341 and assuming the C-terminus at 434, for a combined mol. wt of 36,865 for the fc i fragment. Sedimentation equilibrium ultracentrifugation of Fc i under non-dissociating conditions showed an M n of 36,200 ± 1200, an M W of 36,400 ± 600 and an M z of 37,000 ± 300 g/mole. The best yields of Fc were obtained with a 6-hr digestion and the best yields of fc 1 and Fc' were obtained with digestion for 18hr in phosphate buffer. Digestion in Tris buffer for 18hr gave results similar to the 18-hr digestion in phosphate buffer except the yields of Fc' were less. This fragment may be useful for exploring biological functions of human IgG Fc. In addition, some Fc fragments obtained by papain digestion of human IgG either in phosphate or Tris buffer are not covalently bonded and are probably cleaved on the carboxy-terminal side of the interchain disulfide bonds at Leu 234 or Leu 235, the N-termini for the large polypeptide chain of Fc i. This indicates that, if disulfide bonded Fc fragments are needed, gel filtration under dissociating conditions will be necessary to remove non-covalently bonded Fc.

Hybrids from equine LH: alpha enhances, beta diminishes activity

LH hybrids were prepared by combining eLHα and eLHβ with the corresponding subunits of oLH, pLH and hCG. Recombinants were isolated by gel filtration and assessed by SDS-polyacrylamide gel electrophoresis under both dissociating and non-dissociating conditions. All combinations of subunits produced hybrid LH molecules. Hybrids prepared by combining eLHβ with oLHα, pLHα or hCGα were very inactive in rat radioligand and Leydig cell in vitro bioassays. Hybrids prepared with eLHα were very active in both assays. The greatest potentiating activity was observed when eLHα was combined with pLHβ. The resulting hybrid was 49 times as active as pLH in stimulating steroidogenesis by Leydig cells.

Complexes of viroids with histones and other proteins.

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.

Heterotypic tetramer (A 2D 2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH 1C 1) were used to isolate eytokeratin subunit complexes by solubilization in low salt buffer containing 4 m-urea. Alternatively, the complexes were prepared by solubilixation of total cytoskeletal proteins in 9.5 m-urea or 6 m-guanidinium hydrochloride (Gu · HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 m-urea or 6 m-Gu · HCl and then against buffers containing either 4 m-urea or 2 m-Gu · HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1:1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A ( M r 55,000; isoelectric point in 9.5 m-urea, pH 6.4) and D ( M r 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 m. The complex had a sedimentation coefficient s 25. w of 4.96 S in 2 m-Gu · HCl. Sedimentation equilibrium analysis gave an average M r value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 m-urea or 2 m-Gu · HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional (“diagonal”) gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when eytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics. The data are not compatible with a three-stranded coiled-coil organization. We conclude that hepatic cytokeratin occurs in subunit complexes that are heterotypic tetramers containing two molecules each of polypeptides A and D. We suggest that these complexes represent two-stranded coiled-coils containing one molecule of A and D, which in turn associate into pairs of heterodimers, i.e. tetramers. We propose that these heterodimer pairs represent the basic subunits of polymerization of cytokeratin filaments. The results are discussed in relation to current concepts of molecular arrangements in other types of cytokeratin and in non-keratinous intermediate-sized filaments.

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.

Precursors of storage proteins in Lupinus angustifolius.

The proteins that are synthesized during differentiation and development in the cotyledons of Lupinus angustifolius L. were characterized both in situ and after purification. The proteins present in situ were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and subjected to 'Western'-blot analysis to identify immunologically related polypeptides. The major storage proteins of the lupin, conglutins alpha and beta, were both present in juvenile tissue only as higher Mr precursors. For conglutin beta, a family of at least three polypeptides of Mr 66 000-72 000 accumulated during the earliest phases of protein synthesis in the developing cotyledon (20-28 days after flowering). Later in development each of these polypeptides disappeared and there was the concurrent appearance in the cotyledon of the lower-Mr fragments characteristic of mature conglutin beta. For conglutin alpha, an equivalent family of precursor polypeptides of Mr 60 000-83 000 was detected. Multiple internal sites for proteolytic cleavage of all these precursors appeared to be present. However, processing of the precursors was sufficiently slow to allow them to accumulate to over 50% of total soluble protein in juvenile tissue. The precursors were purified by column chromatography under non-dissociating conditions and shown by ultracentrifugation to be multimeric proteins with Mr values in the range 150 000-200 000.

Characterization of rabbit masseter muscle fibers

Myosins of histochemically distinguishable single fibers of rabbit masseter muscle--type 1, 2A, 2B, and slow fibers--have been characterized by gel electrophoresis under dissociating (sodium dodecyl sulfate) and nondissociating (inorganic pyrophosphate) conditions, and by analysis of peptide maps of the heavy chains following limited proteolytic degradation. Type 2B fibers contain more LC3 homodimer than type 2A fibers; peptide maps of their heavy chain are different although the two myosins comigrate on pyrophosphate gel electrophoresis. Slow fiber myosin migrates more slowly than fast myosin and has a distinct peptide map. Differences were also found among fibers of the same histochemical type but originating in different muscles. In adductor magnus 2B myosin the LC1 + LC3 heterodimer band is the strongest, while in masseter 2B myosin the heterodimer is the weakest. Statistical considerations suggest that in masseter there is a mechanism preferentially forming the homodimers. More work is needed to determine the mechanism by which phenotypical differences occur among various fiber types in the same muscle and between corresponding fiber types in different muscles.

Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide

When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain. The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions. The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates. This behaviour of the light chains-peptide complex mimics that of the intact molecule. The effect of bound light chains and bound actin on the susceptibility to tryptic digestion was studied using scallop S1(+LC) and S1(-LC) (EDTA S1 according to previous nomenclature). The heavy chains of both types of S1 are labile and have two main sites susceptible to proteolysis. Tryptic digestion on site A produces an N-terminal peptide of around 70 000 and a C-terminal 24 000 fragment from S1(+LC) and a 20 000 C-terminal fragment from S1(-LC); the latter is prone to further proteolysis. Thus S1(-LC), produced in the absence of bound regulatory light chain is shorter on the C-terminal end. Proteolysis on site A abolishes actin-activated ATPase activity; the latter is prevented by digesting acto-S1. The rate of tryptic digestion on site B is somewhat slower than on site A; when either S1 is split at this site an N-terminal 63 000 peptide is produced. The corresponding C-terminal peptide can be obtained from acto-S1 when hydrolysis on site A is prevented; this is estimated as around 31 000 derived from S1(+LC) and 28 000 derived from S1(-LC). The results are compared with similar experiments where vertebrate subfragments were digested by trypsin and the possible localization of the light-chain binding peptide in the intact heavy chain is discussed.

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus.

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.

Effects of treadmill running on the properties of guinea-pig myosin

The effects of running training on the structure and function of ventricular myosin of guinea-pigs were studied. No differences in body or heart weights could be detected but the heart-to-body weight relation increased significantly (P less than 0.05) in the trained group. Ca2+ activated and K+ activated ventricular myosin ATPase activities as well as the electrophoretic appearances (SDS-PAGE, pyrophosphate-PAGE) did not change after training. Guinea-pig ventricular myosin in nondissociating conditions showed one band migrating close to rat-V3 isomyosin. The myosins of the trained and untrained animals also showed no immunological difference as determined by the competitive ELISA-test: they both shared antigenic determinants common to rat-V1 isomyosin.

The isomyosins of rabbit middle ear muscles.

Isomyosins of the two middle ear muscles of rabbit have been separated by electrophoresis in non-dissociating conditions. Tensor tympani has four isomyosins called MM1 to MM4 by order of decreasing mobility. Stapedius has two isomyosins, similar to MM1 and MM4. MM1 and MM2 have the same mobility than FM2 and FM3, fast isomyosins observed in psoas. MM4 has the same mobility than IM, a slow isomyosin observed in extensor digitorum longus. MM3 is absent in soleus, psoas or extensor digitorum longus. Slow myosin (SM) of soleus and fast myosin (FM1) of psoas are not observed in middle ear muscles.

The half-cystine content of glutenin subunits of wheat determined by reaction with cystamine dihydrochloride and two-dimensional electrophoresis

The number of half-cystine residues, present in individual subunits of the glutenin fraction of wheat storage proteins that are capable of participating in disulphide interchange reactions, was investigated by treating the seed proteins with a positively-charged disulphide reagent (cystamine di-HCl) in the presence of catalytic amounts of 2-mercaptoethanol and in the absence of denaturing agents such as urea and sodium dodecyl sulphate. Under the conditions used, mixed disulphides are formed between the protein and the reagent, and, for each half-cystine that forms such a mixed disulphide, the protein gains an additional positive charge. After treatment the seed proteins were fractionated by two-dimensional gel electrophoresis using systems that separate proteins on the basis of their isoelectric points. Comparison of the gel patterns of the cystamine di-HCl treated proteins with the patterns of the untreated proteins suggested the following. (a) That the high molecular weight glutenin subunits possess four half-cystine residues capable of forming a mixed disulphide upon reaction with cystamine di-HCl under non-dissociating conditions; it is presumed that these four residues probably participate in interchain disulphide bond formation since the interchain bonds are expected to be the most accessible in the absence of denaturing agents, (b) That at least some of the low molecular weight glutenin subunits also possess ‘reactive’ half-cystine residues, although it was not possible to obtain an estimate of the number of such residues possessed by individual subunits. (c) That several other proteins, which appear not to be gliadins or glutenins, each possesses two ‘reactive’ half-cystine residues. These findings are discussed in relation to the structure of the glutenin aggregates.

Isozymes of myosin in growing and regenerating rat muscles.

Native myosin isozymes of rat muscles have been isolated by electrophoreses in non-dissociating conditions. Their mobilities were measured, using taenia coli myosin as an internal standard and their relative concentrations were determined by computer planimetry of the electrophoretograms. Three isozymes were observed in extensor digitorum longus (EDL), two in soleus (SOL), four in neonatal muscles three days before birth. Regenerates of minced EDL or SOL muscles in adult animals had no native myosin the third day after surgery; they were similar to neonatal muscles 15 days after surgery and to adult muscles 60 days after surgery.

Étude des polypeptides de structure du virus de la maladie lymphokystique des poissons (lymphocystis disease virus) multiplié en culture cellulaire

Summary We report the results of analyses of virion proteins by acrylamide gel electrophoresis. Purity was evaluated in three ways, i. e. by reconstruction experiments, electrophoresis of purified virions artificially mixed with uninfected cell lysate and electrophoresis in non-dissociating conditions of freshly purified virions. Analysis of the polypeptides from purified LDV indicates that after solubilization with SDS-β-mercaptoethanol, 22 polypeptides could be resolved in Coomassie-brillant-blue- or silver-nitrate-stained electrophoretograms as well as in autoradiograms. The molecular weights ranged from 30,700 to 210,000. After chloramin T or lactoperoxidase iodination, we could tentatively localize some proteins in the virions.

Purification and characterization of β-galactoside-binding lectin from chick embryonic skin

Lectin activity was found in tarsometatarsal skin of chick embryo. It was specific for β-linked galactosyl residues and required a thiol-reducing agent for hemagglutination activity. The lectin was extracted from dermis and epidermis (skin) with lactose and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. Examination of their biochemical properties showed that although dermis and epidermis develop from different origins, they contain the same lectin. The apparent subunit M r of lectin was 14 000 and its isoelectric point was 7.0. Under non-dissociating conditions, the lectin exists mainly as a dimer. Radioimmunoassay showed that this skin-type lectin is present in many tissues including skin, muscle, bone, eye, heart, liver and brain at various developmental stages. A wide distribution and a marked change in its content during development strongly suggest that the lectin might have a fundamental role in cellular function, embryonic development and tissue differentiation.

Characterization of a non-functional form of Clq found in patients with a genetically linked deficiency of Clq activity

A genetically defective form of Clq was purified from the sera of patients suffering from an immune complex related disease and who were homozygous for the defect. The defective Clq was haemolytically inactive and did not bind to immune aggregates or IgG-Sepharose. It showed the following similarities to the normal Clq molecule: a high glycine content and the presence of hydroxyproline and hydroxylysine; subunits with apparent mol. wts of 70,000 and 56,000, when examined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions; preferential incorporation of 125I-label into only one of the types of chain present in the molecule, in a manner similar to that found for the C-chain of normal Clq. However, the defective molecule had an apparent mol. wt of approximately 155,000 in non-dissociating conditions, which is approximately one-third of the mol. wt of the normal molecule. Also, the material in the defective molecule preparation which corresponded, on the basis of mol. wt, to the disulphide-linked A-chain-B-chain dimer of normal Clq differed from that found in the normal molecule in that it did not appear to be sensitive to reducing agents. Collagenase and pepsin treatment of specific immunoprecipitates containing the radiolabelled defective molecule indicated that it is, like the normal molecule, composed of collagenous and non-collagenous domains.

Purification of a protein (60K/58K) associated with the Epstein-Barr virus-induced early antigen complex in Raji cells.

A double antibody sandwich ELISA has been established for the detection and quantitation of EBV-associated early antigens (EA) in IUdR-induced Raji cells. The EA complex extracted from Raji cells could be separated by ion exchange chromatography and isoelectric focusing into several components. One EA-associated subspecificity has been purified by DEAE-, CM-, and Blue-Sepharose chromatography followed by isoelectric focusing. The isolated protein has an apparent molecular weight of 240,000 +/- 20,000 daltons under non-dissociating conditions on Sephacryl S-300, an isoelectric point of 4.5, and seems to be composed of two polypeptides of 60,000 and 58,000 daltons as shown by SDS-gel electrophoresis and two-dimensional gel electrophoresis. Preliminary data indicate that the 58,000 polypeptide is generated by limited proteolysis of the 60,000 polypeptide. The EA activity of the isolated protein has been confirmed by the double antibody sandwich ELISA and its reactivity with anti-EA-positive sera in an ELISA for the detection of anti-EA antibodies.