Changes in the acid-soluble nucleotide composition of red cells of cattle at various ages.

DNA polymerase was extracted from HeLa cell mitochondria with high salt concentrations (1M) and Nonidet-P 40 (0.2%). Subsequently the enzyme was purified stepwise by DEAE-cellulose-, phosphocellulose-, hydroxyapatite-Ultrogel-, DNA-cellulose chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme exhibited a molecular weight between 100 000 – 110 000 and was devoid of endonuclease activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this enzyme preparation revealed two protein bands suggesting that the mitochondrial DNA polymerase might consist of two subunits with the molecular weights of 45 000 and 60 000.     

Comparison of tRNA nucleotidyltransferase from Escherichia coli and Lactobacillus acidophilus.

Purification of tRNa nucleotidyltransferase from Lactobacillus acidophilus ATCC 4963 and Escherichia coli MRE 600 by preparative polyacrylamide gel electrophoresis is described. Both enzymes gave a single band on analytical polyacrylamide-gel electroesis and sodium dodecylsulfate gels. Chromatography of the high speed supernatant from Lactobacillus at low salt concentrations gave three enzyme fractions of molecular weights about 45 000, 90 000, and 120 000. At 1M NaCl only the first enzyme fraction was found. Kinetic data for both enzymes are given.     

A large photoreactive particle from Chromatium vinosum chromatophores

Large photoreactive particles from Chromatium vinosum are obtained pure and in high yield by using a mixture of detergents at high ionic strength to dissociate the chromatophore membrane. The particles contain all of the secondary electron acceptor of the chromatophores and about half of the cytochrome. Their content of ubiquinone is greatly enriched as compared with chromatophores. The individual particles have an estimated molecular weight of between 650 000 and 810 000.Gel electrophoresis of the preparation in sodium dodecylsulfate shows polypeptides with molecular weights of 50–45 000, 30 000, 27 000, 22 000 and 12 000. The 50–45 000 components are cytochromes. The 30 000, 27 000 and 22 000 components may be analogous to the triad of polypeptides present in Rhodopseudomonas spheroides reaction centers. The non-cytochrome components are partly soluble in chloroform/methanol.Aggregates of particles appear in these preparations. Electron microscopy of the aggregates demonstrates rectilinear lattices of isodiametric particles, 120 Å in diameter. These sheet-like structures are one unit thick and typically contain 9–16 members. They appear to arise by aggregation during isolation but are probably similar to native aggregates apparent within chromatophores after treatment with detergents at low salt concentration.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

Isoelectric points and molecular weights of salt-extractable ribosomal proteins.

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCL were characterized by acid urea-polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.     

Studies on the relationship between glutathione S-transferase phenotype and bile acid binding by human liver cytosol

The possibility that the GST1 phenotype of human liver cytosol is a determinant of bile salt binding has been investigated by using equilibrium dialysis and gel-exclusion chromatography. Binding of bile salts was non-saturable and whereas the glutathione S-transferases did not appear to be major bile salt binders, other binding components with molecular weights of 35 000 and 11 000 were identified in both fetal and adult cytosols.     

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.     

Purification and characterization of 2-alkenal reductase.

The purification of a 2-alkenal reductase to homogeneity from a rat liver 100 000 times g supernatant is described. Its molecular weight has been determined by Sephadex G-100 chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis before and after reduction with mercaptoethanol and carboxymethylation. The monometric form has a molecular weight of 45 000. It tends to form, to a very small extent, dimeric and trimeric aggregates of molecular weights 90 000 and 135 000. The isoelectric point (IP) was determined to be 6.2 by isoelectric focusing.     

Activation of fibroblast procollagenase by mast cell proteases.

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Isolation of NAD-kinase from pigeon heart

A procedure of isolation of pigeon breast muscle NAD-kinase resulting in a 100--130-fold purification of the enzyme with a total yield of 30--35% is described. The enzyme is electrophoretically homogenous; its molecular weight as determined by SDS-electrophoresis is 45 000. The partially purified preparation contains multiple enzymic forms with molecular weights varying from 45 000 to 270 000, which represent an equilibrious system of structurally different oligomers. At the last purification step, i. e. ion-exchange chromatography, the enzyme loses its ability for oligomerization. Possible causes of disappearance of the enzyme multiple forms during purification are discussed.     

Isolation and characterization of human liver mRNA

Poly(A)-RNA isolated from human liver shows a predominant size in the 18 S region. It can be translated into a broad range of proteins up to molecular weights of more than 100 000 with predominant translation products in the 45 000-55 000 region. Full length cDNA has been transcribed and used for a complexity analysis of this mRNA giving a total sequence complexity of about 7 000 different mRNA species.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Purification and characterization of a collagenous protein secreted by a murine teratocarcinoma-derived cell line

A collagenous protein could be precipitated by (NH₄)₂SO₄ from the culture medium of a murine teratocarcinoma-derived cell line (Ko, C.Y., Johnson, L.D. and Priest, R.E. (1979) Biochim. Biophys. Acta 581, 252–259). Further purification of this protein was achieved by combining DEAE-cellulose chromatography with either CM-cellulose or molecular sieve chromatography. The collagenous polypeptides had subunit molecular weights of 160 000, if determined by molecular sieve chromatography, or 190 000, if determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they are not linked by disulphide bridges. Amino acid composition of this collagen is similar to that of a murine type IV collagen isolated from EHS sarcoma (Timpl et al. (1978) Eur. J. Biochem. 84, 43–52). The most prominent peptides resulting from cleavage of the protein by CNBr had estimated molecular weights of 25 000, 23 000, 11 700 and 9400. Pepsin treatment of this collagen under non-denaturing conditions produced three major fragments having molecular weights of 70 000, 45 000 and 43 000. We conclude that the collagen secreted by the murine teratocarcinoma-derived cell culture is a type IV basement membrane collagen. Therefore, this culture system should provide a continuous source of type IV collagen, which may be used to study the interaction of this collagen with other basement membrane components.     

Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1 b

The NAD-reducing hydrogenase of Nocardia opaca 1 b was found to be a soluble, cytoplasmic enzyme. N. opaca 1 b does not contain an additional membrane-bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 mumol H2 oxidized min-1 mg protein-1. NAD reduction with H2 was completely dependent on the presence of divalent metal ions (Ni2+, Co2+, Mg2+, Mn2+) or of high salt concentrations (0.5-1.5 M). The most specific effect was caused by NiCl2, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl2 was 7.8-8.0. In the absence of Ni2+, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5-fold stimulation of the reactions by 0.1-10 microM NiCl2 was observed. With the most effective electron acceptor, benzyl viologen, a 50-fold higher specific activity was determined than with NAD. The total molecular weight of hydrogenase has been estimated to be 200 000 (gel filtration) and 178 000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non-identical subunits with molecular weights of 64 000, 56 000, 31 000 and 27 000. It was demonstrated by electrophoretic analyses that in the absence of NiCl2 and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64 000 and 31 000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56 000 and 27 000. Immunological comparison of the hydrogenase of N. opaca 1 b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD-linked hydrogenases are partially identical proteins.     

Calcium sensitivity of hybrid complexes of muscle myosin and Physarum proteins.

1. A myosin-actin hybrid complex was used to study actin-associated calcium sensitivity of a "cytoplasmic" actomyosin. The approach should be generally applicable. 2. Low salt extracts of Physarum polycephalum contain actin which remains in solution after centrifugation at 46 000 times g or at 100 000 times g for 1 h. The actin was precipitated by the addition of muscle myosin to the supernatants and detected in the hybrid complex by electron microscopy, sodium dodecyl sulfate gel analysis, super-precipitation and activation of the myosin ATPase activity. Actin was also precipitable from high speed supernatants of brain tissue or platelets. 3. The hybrid complexes from Physarum possessed 1.5-5-fold calcium dependency which could be removed by washing. Reincubation of the washed complex with concentrated wash solution resulted in high calcium sensitivity. On sodium dodecyl sulfate gels, unwashed complexes from Physarum contained high molecular weight material in addition to bands of molecular weights less than actin. The bands in the size range of 39 000 to 18 000 were primarily lost from the Physarum complex concomitantly with loss of calcium dependence. 4. When the Physarum supernatants were made 40 mM in MgCl2, precipitates were formed containing actin which possessed calcium sensitivity which was also lost on washing with low ionic strength solutions. This calcium dependency was partially reversed by the addition of desensitized rabbit actin to the precipitate before assay. 5. Conclusion: calcium regulation of actomyosin in Physarum is mediated primarily by factors that are bound to the actin component. The regulatory factors are soluble in low salt buffers. The molecular weights of the polypeptide chains of several of these factors are similar to those of the troponin polypeptides of striated muscle. In Physarum but not in platelet or brain a prominent polypeptide chain of approx. 55 000 molecular weight also occurs which coprecipitates with the hybrid complex and which is not easily removed.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

On the molecular weight of thiosulfate sulfurtransferase.

Bovine liver thiosulfate sulfurtransferase (rhodanese) (EC 2.8.1.1) HAS BEEN REPORTED TO EXIST IN SOLUTION IN A RAPID, PH-dependent equilibrium between monomeric and dimeric forms of molecular weights 18 500 and 37 000 (Volini, M., DeToma, F. and Westley, J. (1967), J. Biol. Chem. 242, 5220). We have reinvestigated the proposed dissociation using sodium dodecylsulfate-polyacrylamide gel electrophoresis. The smallest rhodanese species observed has a molecular weight around 35 000, which is not reduced by severe denaturing conditions, including alkylation in 8 M guanidine-HCl or dialysis against 2% sodium dodecylsulfate and 5% mercaptoethanol. After limited CNBr cleavage, intermediate products of greater than 18 500 molecular weight are formed. The apparent molecular weight of these intermediate fragments is not changed by addition of mercaptoethanol. The total apparent molecular weights of the CNBr fragments after exhaustive cleavage is approx. 45 000 plus or minus 15 000. These results are not consistent with a monomer molecular weight of approx. 18 500 for thiosulfate sulfurtransferase.