Phosphoprotein Of Molecular Weight Research Articles

Catfish vitellogenin and its messenger RNA are smaller than their chicken and Xenopus counterparts

Injection of male bullheads ( Ameiurus nebulosus) with estradiol induced the production of a major serum phosphoprotein of molecular weight 145 000. This protein was immunoprecipitable by antisera raised against lipovitellin from bullhead eggs and was absent from the serum of control males. Production of this serum protein coincided with changes in the liver mRNA population, which suggested that estradiol had induced the synthesis of additional mRNA sequences in the high-frequency class. Agarose gel electrophoresis in the presence of methyl mercury hydroxide showed that this mRNA population contained at least one species which was not present in the liver of uninjected males. This new RNA was the major polyadenylated species present in the total cellular RNA and its size relative to ribosomal RNAs and locust vitellogenin mRNA was estimated as 5800 nucleotides. When the liver total RNAs were translated in the mRNA-dependent rabbit reticulocyte lysate system the major translation product from the induced fish had the same molecular weight (145 000) as the serum phosphoprotein and was immunoprecipitable by antilipovitellin antisera. This translation product was not coded for by RNA from control fish. These observations are consistent with the induction of vitellogenesis by estradiol as reported in other egg-laying vertebrates and they show that bullhead vitellogenin and its mRNA are significantly smaller than their avian and amphibian counterparts.

Feline sarcoma virus P115-associated protein kinase phosphorylates tyrosine. Identification of a cellular substrate conserved during evolution.

The Gardner strain of feline sarcoma virus (FeSV) has previously been shown to encode as its major translational product, a phosphoprotein of molecular weight (Mr) = 115,000 (P115) with an associated protein kinase activity and probable transforming function. In the present study, we demonstrate that this enzyme specifically phosphorylates tyrosine and that Gardner FeSV-transformed mink and rat cells exhibit constitutively higher levels of phosphotyrosine than nontransformed control cells. In addition, we identify a Mr = 150,000 cellular substrate for the FeSV P115-associated protein kinase activity. This latter protein, designated P150, is expressed in cells of a number of mammalian species including cat, mink, dog, rat, mouse and human, possesses broadly reactive interspecies antigenic determinants, and exhibits binding affinity for FeSV P115. Although P150 exhibits an associated protein kinase activity, this activity differs from the FeSV P115-associated enzyme in that it phosphorylates serine and threonine rather than tyrosine. On the basis of immunologic criteria and tryptic peptide analysis, the possibility that P150 is structurally related to Gardner FeSV P115 is excluded. As a consequence of its affinity for FeSV P115, P150 is efficiently incorporated into FeSV pseudotype virions, providing a source for its purification and preparation of antisera for study of its expression in mammalian cells.

Properties of dissociatively extracted fetal tooth matrix proteins. II. Separation and purification of fetal bovine dentin phosphoprotein.

After an initial 4 M guanidine HCl extraction to remove enamel contaminants, subsequent guanidine HCl/EDTA extraction of fetal bovine molar dentin removes almost all of the high molecular weight phosphoprotein from the tissue in a single step. The fetal bovine dentin phosphoprotein is then purified by a series of ion exchange (DEAE-cellulose or DEAE-Se-phacel) and gel filtration (Sepharose CL-6B) chromatographic steps, all under denaturing elution conditions in 7 M urea (ion exchange) or 4 M guanidine HCl (gel filtration). The final purified phosphoprotein product was chromatographically and electrophoretically homogenous. The apparent molecular weight for this fetal bovine dentin phosphoprotein was approximately 100,000 both by 4 M guanidine HCl gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value is considerably higher than those previously reported for any other dentin phosphoprotein preparation. Roughly half of the total serine residues in the protein were phosphorylated and these residues together with aspartic acid comprised approximately 80% of the total polypeptide backbone. The purified fetal bovine dentin phosphoprotein was also glycosylated, containing approximately 6 galactosamine, 2 to 3 glucosamine, and 2 to 3 sialic acid residues/mol.

Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine.

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.

Effect of ethanol treatment on high molecular weight phosphoprotein phosphatases of rat liver.

When the crude phosphoprotein phosphatase fraction of rat liver cytosol was treated with 80% aqueous ethanol at room temperature, the activity with phosphorylase alpha as substrate was increased by 110%, but those with glycogen synthase D and phosphohistone were decreased by 53 and 34%, respectively. Chromatography of the ethanol-treated fraction on DE-52 revealed that while phosphoprotein phosphatase IA (Mr=69,000) remained to exist even though it was reduced, phosphatases IB (Mr=-300,00) and II (Mr=160,000) were totally replaced by a new phosphatase form with an approximate molecular weight of 35,000. This low molecular weight form has been designated phosphatase III. When partially purified phosphatases IB and II were separately treated with ethanol, they were converted to phosphatase III. These results suggest that phosphoprotein phosphatases IB and II, but IA, contain phosphatase III as a subunit. Phosphatases IB and II, however, must differ in structure since "IB to III" is accompanied by an increase in phosphorylase phosphatase activity much greater than that for "II to III"

The properties of purified low molecular weight phosphoprotein phosphatase from rabbit heart

A simplified procedure for the purification of low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been described from rabbit heart. The enzyme was purified to homogeneity by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34 000. The S20, W value and Stokes radius for the enzyme was 3.35 and 24.0 A(1 A = 0.1 nm), respectively. Using these two values, a molecular weight of 35 000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. Kinetic studies revealed the lowest Km with glycogen synthase D and maximum Vmax for the reaction with phosphorylase a.

Enamel Matrix: Structural Proteins

Cell-free, fetal bovine enamel tissue was examined intact by high resolution. 13C Fourier transform, nuclear magnetic resonance spectroscopy. Two types of protein chains were observed under these conditions, one exhibiting rapid mobility and accounting for approximately two-thirds of the enamel matrix, while the other exhibited restricted or anisotropic segmental motion and accounted for the remaining third of the matrix. Sequential extraction of this fetal enamel under non-degradative conditions with dissociative solvents yielded two biochemically distinct populations of matrix protein. As expected, the bulk of the matrix consisted of proline-rich amelogenins, although the SDS-gel electrophoresis molecular weights for these proteins were somewhat higher than those reported using other extraction methods. Approximately fifteen percent of the total matrix consisted of much higher molecular weight phosphoproteins (46,000-72,000 daltons) whose amino acid composition closely resembled that reported for mature enamel protein. These high molecular weight proteins were tightly bound to the fetal enamel apatite crystallites.

A high molecular weight phosphoprotein in cultured fibroblasts that associates with polymerized tubulin

Microtubule associated proteins (MAPs) are high molecular weight proteins that associate with microtubules during polymerization. This report describes a high molecular weight protein fraction with a molecular weight of approx. 290 000 from cultured mammalian fibroblasts that associates with polymerized rat brain tubulin. This protein(s), which is referred to as f-MAP, is enriched approx. 25-fold in a twice polymerized microtubules when compared with the original cell extract. Polymerization of rat brain extract in the presence of in vivo 32P-labeled fibroblast extract reveals the presence of a 32P-labeled protein in the polymerized pellet with the same electrophoretic mobility as f-MAP. The present study suggests that fibroblasts in culture contain a high molecular weight phosphoprotein with properties and a molecular weight very similar to the MAPs described in mammalian brain.

Cyclic AMP stimulation of membrane phosphorylation and Ca 2+-activated, Mg 2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca 2+-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca 2+-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca 2+-activated ATPase was calcium dependent and maximal at calculated Ca 2+ concentrations of 2.0 μM. Cyclic AMP-dependent Ca 2+-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca 2+-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca 2+-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.

Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH 2[ 32P]O 4 and [ 3H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.

Comparative studies on the structural phosphoproteins of mammalian type C viruses

The major phosphoprotein common to woolly monkey sarcoma virus, gibbon ape lymphosarcoma virus, and type C viruses of the lower mammalian species (mouse, rat, cat), with the exception of the endogenous cat virus (RD-114), is the polypeptide of about 12,000 molecular weight. The protein-phosphate bond in this polypeptide of several viruses is of the phosphoserine variety excepting gibbon ape virus, which contains both phosphoserine and phosphothreonine. The primary phosphoprotein of RD-114 virus and the endogenous baboon type C virus, on the other hand, is the polypeptide of about 15,000 molecular weight which contains phosphothreonine as its phosphoamino acid. A second major phosphoprotein of molecular weight of 10,000 is detected only in viruses genetically related to rat species including those derived from the RPL cell line, from Sprague-Dawley rat embryo cells, and the Kirsten mouse sarcoma virus which was recovered from a mouse erythroblastosis virus after in vivo propagation through rat. These phosphorylated polypeptides of molecular weight 15,000, 12,000, or 10,000 are present in the virion structure in several different but nonrandom phosphorylated states.