Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

Hen oviduct N alpha-acetyltransferase is a ribonucleoprotein having 7 S RNA

Hen oviduct N alpha-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified N alpha-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. N alpha-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureus nuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2 digest of the nucleic acid was composed of four 3'-ribomononucleotides and completely separated from 3'- and 5'-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in N alpha-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that N alpha-acetyltransferase consisted of one molecule of 7 S RNA (Mr 83,000) and two identical Mr 79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of N alpha-acetyltransferase estimated by Sepharose 6B gel filtration.     

Characterization of hepatic lactogen receptor. Subunit composition and hydrodynamic properties

The structure of the membrane-bound and Triton X-100-solubilized female rat liver prolactin receptor has been studied by affinity cross-linking/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and sucrose/H2O and sucrose/D2O density gradient centrifugation. Hydrodynamic characterization revealed that the 125I-human growth hormone receptor-detergent complex represents a molecular species with a Stokes radius of 61 A, a sedimentation coefficient of 5.0 s, and a calculated molecular weight of 158,000. The molecular weight of the receptor was calculated to be 92,000. Three lactogenic hormone-binding species with Mr values of 87,000, 40,000, and 35,000, respectively, were repeatedly found when detergent-solubilized preparations were analyzed using an affinity cross-linking technique. Estrogen treatment of female rats increased the intensity of these bands. Occasionally, an Mr 165,000 hormone-binding species was also found. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies (first dimension, nonreducing; second dimension, reducing) demonstrated that disulfide- and nondisulfide-linked hormone-binding species with Mr values of 40,000 and 35,000 are contained within the Mr 87,000 species. It is concluded that the Triton X-100-solubilized female rat liver prolactin receptor has a molecular weight of about 90,000. This molecular species contains Mr 40,000 and Mr 35,000 hormone-binding subunits. It cannot be determined whether these subunits are combined with each other or with as yet undetected subunit(s) to make up the Mr 90,000 species, or whether each one of these subunits is a proteolytic fragment of the Mr 90,000 species.     

Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase.

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.     

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).     

In vitro synthesis and processing of a precursor to ornithine aminotransferase

Poly(A)+ RNA was isolated from liver-free polysomes of rats maintained on a 60% casein diet by sodium dodecyl sulfate-phenol-chloroform extraction and oligo(dT)-cellulose chromatography. Poly(A)+ RNA translated in a rabbit reticulocyte lysate system produced a polypeptide of 49,000 daltons that was immunoprecipitated by monospecific, affinity-purified IgG antibodies to ornithine aminotransferase (ornithine-oxo acid aminotransferase, EC 2.6.1.13). This polypeptide is 6,000 daltons larger than mature ornithine aminotransferase when electrophoresed on sodium dodecyl sulfate polyacrylamide gels. One-dimensional peptide mapping demonstrated that this 49,000-dalton polypeptide is structurally related to ornithine aminotransferase. Furthermore, it can be processed to a polypeptide of 43,000 daltons by a rat liver mitochondrial fraction. We have concluded that this polypeptide is a precursor to ornithine aminotransferase.     

The subunit structures of soluble and chromatin-bound RNA polymerase II from soybean

DNA-dependent RNA polymerase II is present in two forms, IIa and IIb, in germinating soybean. Form IIa is the dominant form of the enzyme in ungerminated embryos and appears to be a soluble enzyme. Form IIb increases in amount as germination progresses and is tightly bound to the chromatin template. The subunit structures of soybean RNA polymerases IIa and IIb are identical except for the molecular weights of their largest subunits which are 200,000 daltons and 170,000 daltons for IIa and IIb, respectively. The enzymes have seven common subunits: 142,000, 42,000, 26,000, 20,000, 16,000, 15,000, and 14,000 daltons.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.     

Endoplasmic reticulum nuclease. Purification and specificity

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.     

Purification of the DNA-dependent RNA polymerase from the myxobacterium Stigmatella aurantiaca.

The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera.     

Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization

Human transforming growth factor beta 1 (TGF-beta 1) was purified as a latent high Mr complex from human platelets by a six-step procedure. Analysis by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions revealed that the complex was composed of at least three components with apparent Mr values of 13,000, 40,000, and 125,000-160,000. The 13-kDa subunit was part of a disulfide-bonded dimer and was identified by amino acid sequencing as TGF-beta 1. The 40-kDa subunit was identified as the amino-terminal part of the TGF-beta 1 precursor lacking the hydrophobic signal sequence. Partial sequencing of the 125-160-kDa protein revealed that it is distinct from known proteins. The 40-kDa and the 125-160-kDa subunits are linked by disulfide bonds, forming a complex with an apparent Mr of 210,000 on SDS gels under nonreducing conditions. Experiments with partial reduction revealed that each complex contains two 40-kDa components linked by disulfide bonds; in addition, the dimer is disulfide-linked to one 125-160-kDa binding protein. TGF-beta 1 binds noncovalently to the 210-kDa complex, and in bound form, TGF-beta 1 is inactive. Incubations of the latent form of TGF-beta 1 at extreme pH values, in 0.02% SDS or in 8 M urea, lead to activation of TGF-beta 1, whereas the complex was resistant to treatment with 5 M NaCl or heat (3 min at 95 degrees C).     

In Situ Activity of Enzymes on Polyacrylamide Gels of a Deoxyribonucleic Acid-Membrane Fraction Extracted from Pneumococci

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.     

Complexity and polyadenylic acid content of visna virus 60-70S RNA.

The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.     

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.     

Cell-cell recognition in yeast. Molecular nature of the sexual agglutinin from Saccharomyces kluyveri 17-cells

A previous study of Saccharomyces kluyveri 17-cell sexual agglutinin (alpha-agglutinin), solubilized by zymolyase (beta-glucanase) digestion of 17-cells and purified by affinity adsorption to immobilized 16-cell agglutinin (alpha-agglutinin), suggested that the major active component was a glycoprotein of 60,000 daltons and that a minor active component of 40,000 daltons was also present, possibly the result of proteolysis (Pierce, M., and Ballou, C. E. (1983) J. Biol. Chem. 258, 3576-3582). We now show that both of these active components are proteolytic fragments of a larger form with a molecular weight of 134,000, and that the latter is produced by proteolysis of a still larger species with a molecular weight of more than 200,000. Washed 17-cell wall fragments were labeled with 125I and digested with purified protease-free beta-1,3-glucanase, and the solubilized alpha-agglutinin was precipitated with antiserum raised against purified agglutinin containing a mixture of the 60,000- and 134,000-dalton forms. Gel electrophoresis in sodium dodecyl sulfate revealed a radioactive material with Mr greater than 200,000 that, on digestion with zymolyase containing an active protease, was converted sequentially to radioactive components with Mr = 134,000, 60,000, and 40,000.     

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.