Identification and isolation of casein kinase type II from RNA-binding proteins of amphibian oocytes

Protein kinase previously detected in RNA-binding proteins of amphibian oocytes phosphorylates casein far more efficiently than histones to form phosphoserine and phosphothreonine and utilizes both ATP and GTP. Heparin in concentrations below 1 microgram/ml inhibits protein kinase. This allows to relate the enzyme to casein kinases II. Protein kinase was extensively purified (more than 15000-fold) with respect to proteins of ribosome-free extract. The homogeneous enzyme consists of three polypeptide chains (Mr 43,000, 41,000, and 29,000). The 125I-labelled enzyme possessing casein kinase and RNA-binding activities when injected into amphibian oocytes was detected in the particles identical to free cytoplasmic informosomes in terms of their sedimentation properties.     

cAMP-independent protein kinase from amphibian lens: identification, organ distribution and substrates of phosphorylation

Eye lens extracts of the frog Rana temporaria contain a cAMP-independent protein kinase which is quantitatively adsorbed on immobilized RNA at physiological salt concentrations. The enzyme activity is maximal in the lenticular cortex, medium in the epithelium and minimal in the lens nuclei. Crude preparations of RNA-binding protein kinase from the epithelium, cortex and nuclei of the eye lens were prepared by affinity chromatography on poly(U)-Sepharose. It was found that these preparations contain no active forms of phosphatases, ATPases or proteases which may interfere with the results of phosphorylation experiments on exogenous and endogenous substrates. The protein kinase under study catalyzes the binding of phosphate groups to threonine and serine residues in casein molecules, does not phosphorylate histones and utilizes GTP alongside with ATP as phosphate donors. Heparin and RNA used at low concentrations inhibit the protein kinase activity. The data obtained allow the identification of lenticular RNA-binding protein kinase(s) as a casein kinase type II. It was shown that incubation of RNA-binding proteins from epithelium and lenticular cortex with [gamma-32P]ATP results in the label incorporation into six to seven polypeptide chains with Mr of 27-130 kDa. Poly(U) and heparin inhibit the self-phosphorylation reaction, cAMP has no stimulating effect on this process, while Ca2+ ions inhibit the self-phosphorylation of RNA-binding proteins.     

Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells.

One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.     

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.     

The search for proteins with immunochemical affinity to plant stress proteins at cold-adapted endemic Baikal fishes

The search for proteins with immunochemical affinity to plant stress proteins in endemic Baikal fishes shows the presence of proteins, immunochemically related to plant heat-stabile proteins and plant uncoupling protein CSP 310. Western blotting showed that among the native cytoplasmic proteins of endemic Baikal fishes there are proteins immunochemically related to heat-stabile plant proteins with molecular weights about 480, 200-290, 150, 140 and about 90-100kD. SDS-electrophoresis showed the presence of polypeptides with molecular weights 23, 17 and 14kD in all species investigated and an additional 35kD polypeptide in Cottocomephorus grewingki. The search for polypeptides with immunochemical affinity to plant stress uncoupling protein CSP 310 in endemic Baikal fishes shows the presence of a 14kD polypeptide, immunochemically related to it.     

Molecular structure of the beta-adrenergic receptor

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.     

The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.     

Intraspecies heterogeneity of epidermal keratins isolated from bovine hoof and snout.

The alpha-keratins, the principal components of the tonafilaments, were extracted, characterized and compared in bovine hoof and snout epidermis. The alpha-fibrous proteins of these tissues are similar with respect to their molecular weights, amino acid composition and percentage of helical structure. However, distinct differences in the polypeptides comprising these proteins were observed. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these proteins consistently showed that the polypeptide chain in snout, designated as band B (mol.wt. 67,000), was completely absent from hoof preparations. This was confirmed with several alternative preparative procedures. The peptides produced by digestion of the intact keratins from hoof and snout with CNBr were distinctly different. Finally, digestion of keratins from hoof and snout with trypsin yielded products that differed in size and resistance to further digestion. Thus, in addition to the interspecies polypeptide heterogeneity documented in the literature, this report establishes the intraspecies heterogeneity of keratins and suggests that these differences are due to either the expression of different gene products or differences in post-translational modifications in these two tissues.     

Two-dimensional electrophoretic analyses of proteins synthesized during differentiation of 3T3-L1 preadipocytes.

The changes in protein composition and cell surface proteins that occur during the adipocyte conversion of 3T3-L1 preadipocytes were monitored by two-dimensional polyacrylamide gel electrophoresis folowing incubation of cells with [35S]methionine for periods of 3 and 24 h. Alterations in the biosynthesis of more than 30 cytoplasmic proteins, 9 non-histone, chromosome-associated proteins, and 24 membrane proteins, were detected. Although the methodological limitations of the electrophoretic systems employed result in an underestimate of the total number of differences, the alterations observed exceed the enzyme changes known to occur during differentiation of these cells. One major alteration occurring during differentiation is a decrease in the content of a protein whose position following two-dimensional electrophoresis tentatively identified it as actin. A fall in actin content accompanying adipocyte conversion was confirmed by direct analysis of the DNase 1 inhibitory activity in homogenates prepared from cells during the course of differentiation. Studies of cell surface proteins by lactoperoxidase-catalyzed iodination reveal a number of changes during differentiation including an increase in a polypeptide(s) in the molecular weight range of 16,500 to 18,500, a decrease in at least four proteins of molecular weights greater than 100,000, and in a protein of molecular weight 95,000.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.     

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.     

Purification and partial characterization of two cyclic amp-independent protein kinases from AH-66 hepatoma cells

Two cyclic AMP-independent protein kinases (CK 1 and CK 2) in the cytosol fraction of AH-66 hepatoma cells have been purified to homogeneity. CK 1 and CK 2 had molecular weights of 36,000 and 340,000, respectively, as determined by gel filtration. CK 1 consists of a single polypeptide and CK 2 is composed of two subunits of 42,000 and 26,000 molecular weights. CK 1 and the 26,000 molecular weight subunit of CK 2 were autophosphorylated. Both enzymes catalyzed the phosphorylation of cytosolic proteins including those with molecular weights of 125,000, 95,000, 56,000, and 40,000.     

beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions.

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).     

beta-Bungarotoxin. The binding of [3H]pyridoxylated beta-bungarotoxin to a high-molecular-weight protein receptor.

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).     

Liver protein synthesis. Molecular weight distribution of pulse-labeled polypeptide chains in normal and thyroidectomized rats.

Equations are presented for determination of elongation rate in vivo for a heterogenous population of polypeptide chain molecular weights. The distribution of pulse-labeled polypeptide chains in rat liver deoxycholate-soluble protein has been obtained by sodium dodecyl sulfate-gel electrophoresis and used to compute a theoretical curve for determination of synthesis time of a 50000 mol. wt. polypeptide chain (tc50). Values of tc50 for normal and thyro-parathyroidectomized Long-Evans male rats were 1.2 and 1.75 min, respectively, representing protein synthetic rates of about 7.5 and 5.1 mg protein/g liver/h. No difference in the molecular weight profile of liver polypeptide chains on the basis of labeling or Amido-black staining was observed between the two groups. The distributions of radioactivity before and after secretion of labeled plasma protein are compared. The role of protein-synthetic rate in the changing enzyme levels associated with thyroid hormone is discussed.     

CYTOPLASMIC SYNTHESIS OF NUCLEAR PROTEINS : Kinetics of Accumulation of Radioactive Proteins in Various Cell Fractions after Brief Pulses

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.     

Isolation of a high-molecular-weight high-mobility-group-type non-histone protein from hen oviduct.

An organ-specific non-histone protein, with a mol.wt. of 95,000, was isolated from hen oviduct. This protein consists of approximately equal amounts of acidic and basic amino acids and has an isolectric point of 7.4. On the basis of its known characteristics, this protein is similar to the high-mobility-group proteins observed in other tissues.