Purification and immunological analysis of RNA polymerase II from Caenorhabditis elegans

We describe a rapid procedure for obtaining highly purified RNA polymerase II from the nematode Caenorhabditis elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of three large polypeptides (molecular weights 200,000, 175,000, and 135,000) and eight smaller polypeptides (molecular weights 29,500, 20,000, 16,000, 15,000, 13,000, 11,500, 10,500, and 9,500). As observed for the analogous enzyme from other organisms, the 175,000 polypeptide (II175) appeared to be a degraded form of the 200,000 polypeptide (II200). The structure of nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Four of its larger subunits shared antigenicity with Drosophila RNA polymerase II. Antibody raised against purified RNA polymerase II reacted with several enzyme subunits in "Western" blots of purified polymerase and impure enzyme fractions. Immunofluorescence staining was used to visualize RNA polymerase II in the nuclei of a nematode squash preparation and the nucleoplasm of cultured mammalian cells.     

Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12.

Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.     

Properties of Arylamidase from Aspergillus oryzae

α-Amylase-like proteins were synthesized in a heterologous cell-free protein synthesizing system prepared from Escherichia coli. The proteins were precipitable with anti-α-amylase serum and detected only when RNA extracted from α-amylase producing Bacillus subtilis cells was used as messenger. The in vitro α-amylase-like products seemed to consist of two components having molecular weights of 30,000 and 13,000.     

Drop weight variation in an automated collection procedure and its relationship to apparent surface tension

A drop counter-regulated fraction collector yields samples containing equal numbers of drops. Such fractions vary slightly in weight depending on experimental conditions such as surface tension. Provided that variables such as flow rate and eluate density remain constant, apparent surface tension may be estimated directly from the weights of eluate fractions obtained from gel filtration experiments.The detergents sodium cholate and sodium lauryl sulphate significantly decreased drop weights in this system. Following gel filtration on Sepharose 4B, sodium cholate eluted in the fractions containing low molecular weight material. It eluted in the same position when pre-mixed with human plasma.Normal plasma was found to contain two surface tension-reducing components with apparent molecular weight of 3· 10⁶ and 1·10⁵. The apparent surface tension of whole human plasma was found to be time dependent and decreased as the flow rate was reduced.     

Characterization of an entomopoxvirus of the lesser cornstalk borer (Elasmopalpus lignosellus)

A biochemical and limited morphological characterization of an entomopoxvirus infecting the lesser cornstalk borer, Elasmopalpus lignosellus, was made. The oval virions measure 270 × 200 nm and the spheroids average 1.5 μm in diameter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis elucidated 32 structural polypeptides with molecular weights ranging from 13,000 to 145,000. The viral genome was examined with the restriction endonuclease EcoRI. Gel electrophoresis of the digested DNA yielded 26 bands and a total molecular weight of 140.8 × 10⁶.     

Effect of Plumbago zeylanica root powder induced preimplantationary loss and abortion on uterine luminal proteins in albino rats

P. zeylanica treatment during first 7 days of pregnancy abolished uterine proteins of 13,000, 19,000 and 26,000 and 75,000 Da molecular weights resulting in preimplantationary loss. Proteins having molecular weights 55,000 and 65,000 Da were absent in aborted rats, that were given P. zeylanica root powder since day 6 to day 17 of pregnancy. The results suggest that proteins having molecular weights 13,000, 19,000, 26,000 and 75,000 Da influence the implantation and proteins of 55,000 and 65,000 Da are required for the maintenance of the pregnancy.     

The hyperactivity of hamster fibroblast lysosomal enzymes after endocytosis of sucrose.

The sialoglycoproteins of the human erythrocyte membrane can be separated into at least eight periodic acid Schiff-positive components using sodium dodecyl sulfate gel electrophoresis in the discontinuous buffer system of Laemmli. All eight components can also be labeled by mild periodate oxidation followed by reduction with NaB³H₄. Periodic acid Schiff 2 can be resolved into at least three distinct components. Two sialoglycoproteins, with apparent molecular weights of 35,000 and 27,000 do not appear to be labeled by lactoperoxidase-catalyzed iodination at the external membrane surface.     

High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.     

Insulin enhances protein phosphorylation in isolated hepatocytes by inhibiting an amiloride sensitive phosphatase

The effects of amiloride on basal and hormone-stimulated protein phosphorylation were studied in isolated rat hepatocytes labeled with ³²P-phosphate. Two types of effect on basal phosphorylation were detected: 1. an increase in labeling of two proteins with molecular weights 93,000 and 18,000; 2. a decrease in labeling of proteins with molecular weights 46,000, 34,000, 22,000 and 13,000. All these effects were dose-dependent (maximum with 0,8-to 1 mM) and reached a maximum after 30 to 40 min treatment of the cells with the drug. Amiloride inhibited specifically all insulin effects whereas glucagon specific effects were largely unaffected. In pulse-chase experiments, amiloride increased and insulin decreased the rate of dephosphorylation of the same proteins (Mr 46,000, 34,000 and 22,000). The data support the conclusion that in hepatocytes insulin increases the degree of phosphorylation of proteins by inhibiting an amiloride-sensitive phosphatase.     

Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies.

The bovine leukemia virus (BLV) was purified from a chronically infected fetal lamb kidney cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this virus revealed the presence of eight distinguishable viral components with molecular weights ranging from 80,000 to 11,000. The major component is a non-glycosylated protein having a molecular weight of 24,000 (p24). At least three heavier polypeptides were found, one of them representing a glycoprotein (gp 60). In addition, four minor polypeptides with respective molecular weights of 19,000, 16,000, 13,000, and 11,000 were identified. In a complement fixation assay using naturally occurring antibodies of a leukemic cow, four polypeptides, which included gp 60, p35, p24, and p16, were found to be reactive.     

Subunits of Limnodrilus erythrocruorin.

The dissociation of the erythrocruorin of the oligochaete $\text{Limnodrilus gotoi}$ was investigated using polyacrylamide gel electrophoresis at neutral pH. In the presence of 0.1% SDS, the erythrocruorin dissociated into five subunits possessing molecular weights of 13,000 (1), 20,000 (2), 23,000 (3), 25,000 (4) and 47,000 (5). In the presence of SDS and mercaptoethanol, the erythrocruorin dissociated into two subunits, whose molecular weights were 13,000 (I) and 28,000 (II). Subunit I accounts for 70–80% of the whole molecule. SDS electrophoresis of the isolated subunits 1 through 5 in the presence of mercaptoethanol showed that subunit I was derived from both subunits 1 and 5, while subunit II was derived from subunits 2–4. These results suggest that $\text{Limnodrilus}$ erythrocruorin consists of at least five polypeptide chains: two chains of 13,000 and three chains of 28,000.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Analysis of Histones from the Endosymbiont Nucleus of a Binucleate Dinoflagellate1,2

Histones were prepared from chromatin of the eukaryotic (endosymbiont) nucleus of Peridinium balticum (Levander) Lemmermann. The amino acid composition of whole histone was rich in lysine and similar to that of Olisthodiscus luteus and Euglena gracilis. Electropheretic analysis of these proteins in acidic-urea disc gels revealed four major bands: one with a mobility slightly lower than that of calf thymus HI; and three others which comigrated with calf H2B, H2A, and H4, respectively. The low mobility band was soluble in 5% perchloric acid and was sensitive to FeCl₃ destaining. Electrophoresis in slab gels containing 0.1% SDS revealed five major components, with approximate molecular weights of 23,000, 20,000, 15,000, 13,000, and 11,000, respectively. The 15,000 and 11,000 dalton histones had mobilities identical to those of calf H3 and H4, respectively. The two highest molecular weight components were soluble in 5% perchloric acid. No bands were found to comigrate with calf H2A or H2B but a band was present that migrated to a position intermediate between calf H2A and H4 (13,000 dalton histone). Two-dimensional gels consisting of acidic-urea gels in the first dimension and SDS gels in the second dimension revealed that the 20,000 dalton component and the 13,000 dalton component are not resolved in the acidic-urea gel. As a working hypothesis, it is suggested that two of the five bands seen in SDS gels represent an H1-like doublet, and two are analagous to H3 and H4, respectively. The remaining histone may replace H2A and H2B.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

Cell surface constituents of sarcoma 180 ascites tumor cells

The cell surface protein components of Sarcoma 180 ascites tumor cells have been investigated by a combination of plasma membrane isolation techniques and lactoperoxidase iodination. For plasma membrane isolation cells were homogenized in the presence or absence of Zn²⁺ and fractionated by sucrose density gradient centrifugation or a two-phase partition to give large membrane fragments or membrane envelopes. Membrane purification was monitored by phase contrast microscopy and chemical and enzyme marker assays. The membrane preparations were analyzed by acrylamide gel electrophoresis in sodium dodecylsulfate. Each preparation showed a common protein pattern of about 15 bands ranging in molecular weights from 33 000 to >300000. Two carbohydrate-containing bands were also present in all preparations. Membranes prepared with Zn²⁺ were much less fragmented and showed much greater amounts of three high molecular weight components than those prepared in the absence of Zn²⁺. This might suggest a role for these components in membrane stabilization.The tumor cells were also subjected to iodination with lactoperoxidase, followed by membrane isolation and acrylamide gel electrophoresis in sodium dodecylsulfate in order to identify polypeptides accessible to the cell surface. The major radioactive band coincided with the major carbohydrate-containing band, presumably a surface glycoprotein. A second carbohydrate-containing band showed variable labeling behavior between different cell preparations. This material had a high molecular weight, as indicated by both acrylamide gel electrophoresis and gel permeation chromatography in dodecylsulfate. Several other components are labeled to a lesser extent in the intact cell.     

Protein components of two different regions of an intestinal epithelial cell membrane: Regional singularities

The two different regions of the plasma membrane, i.e. apical and basolateral membranes, of intestinal epithelial cells were analyzed as to their proten components. They showed very contrasting profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apical membranes possessed several major components with apparent molecular weights larger than 108 000, most of which were also periodic acid-Schiff reagent positive. In contrast, there were no protein components with corresponding molecular weights in the basolateral membrane. The electrophoretic profile of the latter was strinkingly simple. The dominant band was assigned a molecular weight of 101 000 and was periodic acid-Schiff reagent negative. No major components were shared by the two membranes.     

Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 ± 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.     

ELECTROPHORETIC CHARACTERIZATION OF LEUCINE-, GLUCOSAMINE- AND FUCOSE-LABELLED PROTEINS RAPIDLY TRANSPORTED IN FROG SCIATIC NERVE

—[³H]Leucine, [³H]glucosamine and [³H]fucose were incorporated in vitro into proteins in frog sciatic ganglia and subsequently transported at a rapid rate along the sciatic nerve towards a ligature, in front of which they accumulated. The synthesis of transported fucose-labelled proteins is closely linked to protein synthesis but is not dependent on RNA synthesis, as judged by effects after incubation for 17 h in the presence of cycloheximide and actinomycin D. Labelled ganglionic as well as transported material were solubilized in sodium dodecyl sulphate and characterized by polyacrylamide gel electrophoresis. The bulk of ganglionic proteins, labelled with any of the precursors used, had molecular weights exceeding 40,000. The radioactivity patterns of leucine- and glucosamine-labelled ganglionic proteins showed similarities with dominant peaks corresponding to molecular weights of about 75,000 and 50,000. The last peak was almost lacking in fucose-labelled ganglionic components. Leucine- and glucosamine labelled-transported proteins exhibited characteristic and similar electrophoretic distributions in contrast to the pattern of fucose-labelled nerve proteins, which was more polydisperse. The most conspicious nerve proteins corresponded to molecular weights of about 75,000 and 18,000. There was a remarkable agreement in the profile of leucine-labelled transported nerve proteins and fucose-labelled ganglionic proteins. In the light of these observations the possibility that glycoproteins constitute a large part of rapidly transported proteins will be discussed.     

Proteins of the Group B Arbovirus Kunjin

Purified Kunjin virus was disrupted with sodium dodecyl sulfate, urea, and mercaptoethanol or acetic acid. Electrophoresis on 7.5% polyacrylamide gel separated four proteins of high (120,000), intermediate (65,000) and low (18,000 and 13,000) molecular weights. A "core" particle was obtained by degradation of the virion with deoxycholate at 0 C; it contained the viral ribonucleic acid and the two small structural proteins. The "envelope" material released from around the core was identified with the most prominent (intermediate) protein seen in electropherograms of virion proteins. In addition to the structural proteins, at least three additional proteins (specified by virus infection) were found in the cytoplasm. The cytoplasmic proteins associated with Kunjin virus differed in their electrophoretic profile from those associated with infection by the related Murray Valley encephalitis virus.