Purification and some properties of the phosphoprotein from rat incisors.

The phosphoprotein of rat incisors has been purified by successive gel and ion-exchange chromatography. The product gave a single band on polyacrylamide gel electrophoresis and contained approximately 34% phosphoserine and 32% aspartic acid. Alkaline elimination experiments showed all the phosphate to be present as phosphoserine. Ultraviolet spectra in the presence or absence of ATP showed that the phosphoprotein did not contain an nucleotide moiety as suggested by Veis, A., Spector, A. R. and Zamoscianyk, H. ((1972) Biochim. Biophys. Acta 257, 404-413) for bovine dentin phosphoprotein.     

Differences between enamel-related and cementum-related dentin in the rat incisor with special emphasis on the phosphoproteins

In order to determine whether qualitative and quantitative differences exist between the non-collagenous proteins of crown and root dentin, rat incisors were separated into their enamel- and cementum-related dentin portions (ERD and CRD, respectively). Isolation of the mineral-bound proteins was performed under nondegradative conditions. Analytical procedures included DEAE-chromatography on high pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and determination of phosphate, protein, and hydroxyproline. The results have shown that considerable differences exist among the two dentins with respect to the quantity of the various phosphoproteins. For this group of proteins as a whole, the ERD contains about 2 times the amount of organic phosphate found in the CRD and about 1.4 times the amount of protein. The content of higher phosphorylated phosphoproteins was about 4 times higher in the ERD than in the CRD, whereas the reverse was shown for the lower phosphorylated phosphoproteins. All differences were found to be statistically significant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that while the ERD contains phosphoproteins with an apparent molecular mass of 98 kDa, the CRD contains two classes of phosphoproteins one of 98 and one of 88 kDa. The relevance of the observed differences in phosphoprotein distribution is discussed in relation to their possible role in mineralization.     

Improvement in the resolution of human sperm protamines by use of iodoacetamide as alkylating agent.

By use of the neutral alkylating agent iodo [14C1] acetamide instead of ethylene imine or iodoacetate, the resolution of human protamines on gel electrophoresis and ion-exchange chromatography has been improved. Using 20-cm gels, human protamines may be fractionated into seven bands, including the two chromatographically distinct forms of HP1 and a hitherto undetected component HP4. On ion-exchange chromatography, HP2 and the two forms of HP1 may be isolated in sufficient purity for sequence analysis.     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

Partially dephosphorylated phosphopeptide AcSer(P)-Ser(P)-Ser(P) is an excellent substrate for casein kinase-2

The synthetic phosphopeptide AcSer(P)-Ser(P)-Ser(P), reproducing a recurrent feature of casein and other phosphoproteins, once partially dephosphorylated by acid phosphatase, serves as an efficient substrate for casein kinase-2. Previous dephosphorylation beyond 30% hinders subsequent phosphorylation and the entirely dephosphorylated peptide is not a substrate at all. The kinetic constants of the partially dephosphorylated phosphopeptide are much more favourable than those of the synthetic peptides SEEEAA, SSEE and SEE, the latter one being totally inert. Optimal phosphorylation occurs at pH values that ensure complete ionization of the phosphoseryl side chains. These data provide incontrovertible demonstration that phosphoserine can replace carboxylic amino acids as specificity determinant for CK-2, being more effective than glutamic acid itself.     

Phosphorylation of calf thymus RNA polymerase II by nuclear cyclic 3',5'-AMP-independent protein kinase.

Nucleoplasmic RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from calfthymus is phosphorylated by homologous cyclic AMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under non-denaturing conditions revealed that both forms of the enzyme were phosphorylated. Polyacrylamide gel electrophoresis of the 32P-labeled RNA polymerase II under denaturing conditions showed that the 25 000 dalton subunit was the phosphate acceptor subunit. Partial acid hydrolysis of the 32P-labeled RNA polymerase II followed by ion-exchange chromatography revealed serine and threonine as the [32P]phosphate acceptor amino acids. Phosphorylation of the RNA polymerase II was accompanied by a stimulation of enzymatic activity and was dependent upon the presence of ATP.     

Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.     

Separation and partial characterization of guinea-pig caseins.

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii.

In order to determine whether microtubular proteins of flagellar axonemes were phosphorylated, cells of Chlamydomonas reinhardtii were grown in medium containing [32P]orthophosphate for several generations. Only one (alpha subunit) of the two tubulin polypeptides separated by Na dodecyl-SO4-polyacrylamide gel electrophoresis appeared labeled, as detected by autoradiography of the dried gel. 3H- and 32P-labeled alpha tubulin subunit purified by preparative Na dodecyl-SO4-polyacrylamide gel electrophoresis and Na dodecyl-SO4-hydroxyapatite chromatography contained about 0.2 mol of phosphate per mol of polypeptide. Upon partial acid hydrolysis, radioactivity could be accounted for as serine and threonine phosphate. By altering the conditions of the Na dodecyl-SO4-polyacrylamide gel electrophoresis is was possible to resolve the purified alpha-tubulin subunit into five or more components: a major band comprising approximately 65% of the total mass, not phosphorylated, and four or more minor bands comprising together 35% of the mass. Among the minor components at least two were phosphorylated.     

Studies on Stigma Pellicle Glycoproteins of Raphanus sativus L.

Stigma surface diffusates of Raphanus sativus were subjected to polyacrylamide gel electrophoresis. There appeared protein bands, one of which gave positive PAS reaction, indicating it was glycoprotein. SDS polyacrylamide gel electrophoresis showed that the stigma diffusates contained many protein bands. By comparing their mobilities with those of standard proteins of known molecular weights, the molecular weights of some of the major fractions were estimated to be 15000, 30000—46000 and 70000 daltons. After Schiff reagent staining two main glycoprotein fractions appeared on the SDS gel electrophoretic pattern. Their molecular weights were estimated to be lower than 15000 and higher than 100000 daltons. By using acrylamide gel isoelectric focusing method, it was found that the stigma surface diffusates contained an acidic glycoprotein with pH of about 3.7. The amino acid composition of the purified stigma glycoprotein was determined with amino acid analyzer. Glycine, glutamic acid, serine, aspartic acid were some of the predominant amino acids. The diffusates were analysed by gasliquid chromatography for sugars. Results showed that the carbohydrate fraction of the glycoprotein consisted of arabinose 17.3%; galactose 19.1%, xylose 8.1%, mannose 5.4%, glucose 23.7%, rhamnose and/or fucose 26.4%. In the stigma surface diffusates of Raphanus sativus, the content of protein was estimated to be 16% and that carbohydrate was 11%.     

Isolation and partial characterization of rabbit plasma alpha1-antitrypsin.

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei.

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.     

Partial characterization of oat and rye phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (sigma(200)) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a sigma(200) value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

Proteoglycans of Microsomal Fraction of Mouse Mastocytoma

Proteoglycans have been isolated from a microsomal fraction of a mouse mastocytoma by procedures which should minimize alteration of the original protein-polysr, ccharide molecule. The methods used include in vivo labeling of sulfate-containing proteoglycans with³⁵S-sulfate, centrifugation of the tumor homogenate at 105, 000 g, solubilization of the microsomal pellet using sodium dodecyl sulfate, cetylpyridinium fractionation, DEAE-cellulose column chromatography and Geon resin electrophoresis. Two major sulfated proteoglycan fractions were obtained. The analytical data obtained were interpreted to indicate that one of these fractions contained keratan sulfate-like material (KSP), the other a heparin-like polymer (HP). KSP was found to contain sialic acid. The protein content of KSP was considerably higher than that of HP. Results of amino acid analysis indicate that glutamic acid and leucine were predominant in KSP, but serine and glycine in HP. Both KSP and HP were found to be homogeneous when examined using acrylamide gel and cellulose acetate electrophoresis, and HP using Geon resin electrophoresis.     

Molecular characterization of limulin, a sialic acid binding lectin from the hemolymph of the horseshoe crab, Limulus polyphemus.

The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of Limulus polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol, two major protein bands were observed. These two bands, subsequently found to contain carbohydrate as well, corresponded to molecular weights of 25 000 and 27 000. Amino acid sequence analyses were performed on both the intact protein and isolated cyanogen bromide fragments. The following primary structural features were noted in the amino-terminal region of limulin: (1) the absence of histidine and alanine from the NH2-terminal 50 residues; (2) the presence of five of the total eight prolines of the molecule between positions 13 and 30; and (3) a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The resultsof cyanogen bromide cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58 respectively. No sequence heterogeneity was observed in this study. While it is quite possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.