Synthesis and structure of proteoglycan core protein

Studies of the structure and synthesis of cartilage proteoglycan core protein have been carried out. Deglycosylation of completed, secreted proteoglycan by HF-pyridine treatment yielded an intact homogeneous core protein of approximately 210,000 daltons, with a blocked amino-terminus. Greater than 95% of chondroitin sulfate chains and 80% of N- and O-linked oligosaccharides were removed by the procedure, which made the product an excellent xylosyltransferase acceptor. Little alteration of core protein structure occurred during the HF-pyridine treatment as shown by complete immunoreactivity with antiserums prepared against hyaluronidase-digested proteoglycan. In other studies, the initially synthesized precursor for proteoglycan core protein was found to be approximately 376,000 daltons and localized to the rough membrane fractions. This precursor already contained N-linked oligosaccharides, and was also able to accept xylose, thereby initiating chondroitin sulfate chains. The precursor was translocated intact in an energy-dependent manner to smooth membrane-Golgi fractions where further processing of high mannose type of oligosaccharides and addition of glycosaminoglycan chains occurred. The subcellular distribution pattern of the chondroitin sulfate-synthesizing enzymes corroborated the proposed topological modifications of the proteoglycan core protein precursor.     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.     

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.     

Appearance of a proteoglycan in developing sea urchin embryos

It was shown that a proteoglycan is synthetized by embryos of a Japanese sea urchin, Hemicentrotus pulcherrimus. This proteoglycan appears as a single peak on sucrose density gradient ultracentrifugation throughout the development. About half of the mucopolysaccharide moiety in this proteoglycan was found to be dermatan sulphate and the rest to be chondroitinase-resistant mucopolysaccharides.Evidence is presented to show that both types of mucopolysaccharide do not exist in a free form but reside as an integral part of the proteoglycan. The linkage between mucopolysaccharide and protein moieties of the proteoglycan appeared not be an O-glucosidic bond, which is common among other proteoglycans such as proteochodroitin sulphate and proteodermatan sulphate.     

Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.     

Dipetalonema viteae: isoelectric focusing and immunochemical studies on somatic extracts of adult worms and microfilariae.

Isoelectric focusing (IEF) of a somatic extract of adult worms (SEAW) yielded nine fractions. Most of the applied protein antigen was recovered in five fractions with pI values of 5.2, 4.4, 4.3, 4.0, and 3.3, respectively. The nine IEF fractions of SEAW gave a total of 37 bands following electrophoresis on polyacrylamide gel (PAGE). IEF fractionation of a somatic extract of microfilariae (SEM) yielded nine fractions. Two fractions with pI values of 4.4 and 3.2, respectively, contained most of the applied protein. The nine IEF fractions of SEM gave a total of 32 bands following PAGE. Crossed immunoelectrophoresis (CI) of SEM and SEAW against serum from hyperinfected hamsters yielded five and eight precipitation peaks, respectively. CI of SEM and SEAW against their homologous rabbit antisera gave 7 and 10 major precipitation peaks, respectively. Immunodiffusion of the nine IEF fractions from both SEM and SEAW against both their homologous and heterologous rabbit antisera indicated three and four precipitin bands peculiar only to SEM and SEAW, respectively. The remaining 23 bands from both preparations showed lines of identity.     

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans

Proteoglycans from bovine tracheal cartilage were digested with trypsin and chymotrypsin by procedures similar to those described by Mathews (Biochem. J.125, 37 (1971)). Chondroitin sulfate-peptide fragments in the digest were precipitated with cetylpyridinium chloride and subsequently fractionated on a preparative Sepharose 6B column. The fragments, which emerged from the column as a broad peak, were divided into five fractions. Rechromatography of these fractions on an analytical Sepharose 6B column indicated that they had K_av values from 0.17 (fraction 1) to 0.62 (fraction 5). The weight average molecular weight values obtained by meniscus depletion equilibrium centrifugation were 193,000, 126,000, 80,000, 46,000, and 23,000 for fractions 1 to 5, respectively. Values for the molecular weights and for the limiting viscosity numbers, [η], of the fractions were used to determine estimates for α of 0.40–0.46 and for K of 0.43–0.88 in the equation [η] = K·M_v^α. These values for α are consistent with a branched structure for the chondroitin sulfate fractions. Papain digests of each of the fractions were chromatographed on Sephadex G-200. The observed distributions of the monomer chains released by this protease were almost the same for each sample, which indicates that the individual chondroitin sulfate chains in all of the original fractions had nearly the same average molecular weights. The data in sum indicate that peptide fragments which contain from 1 to 8 polysaccharide chains are released when the proteoglycans are digested with trypsin-chymotrypsin.Analytical data indicated that all fractions contained 3–11% of their polysaccharide as keratan sulfate. This indicates either that about 50% of the keratan sulfate chains in the original proteoglycan molecules are located in close proximity to the chondroitin sulfate chains or that some peptides contain large numbers of keratan sulfate chains. Proteoglycan preparations which differed by a factor of about 6 in their ratio of chondroitin sulfate to protein yielded very similar elution patterns on Sepharose 6B after trypsin-chymotrypsin digestion.     

The composition of cartilage proteoglycans. An investigation using high-and low-ionic-strength extraction procedures

1. A proteoglycan fraction (the proteoglycan subunit fraction) was prepared from extracts, with 0.15m-KCl (low-ionic-strength) and 0.5m-LaCl(3), 2.0m-CaCl(2) and 4.0m-guanidinium chloride (high-ionic-strength), of bovine nasal cartilage by equilibrium-density-gradient centrifugation, essentially as described by Hascall & Sajdera (1969). 2. The use of different centrifugation times showed that near-equilibrium conditions were reached by 48h for the fractions prepared from the high-ionic-strength extracts. The fraction isolated from the low-ionic-strength extract required a longer centrifugation time to reach equilibrium conditions. 3. The composition of the proteoglycan fractions from the various extracts was compared by analyses of their carbohydrate and amino acid contents. Difference indices were calculated from the amino acid analysis to compare the degree of compositional relationship between the protein components of the proteoglycans. 4. Small compositional differences were found between the proteoglycans isolated from the various high-ionic-strength extracts. The protein content of the fractions from the CaCl(2) extract and the guanidinium chloride extract showed the greatest difference in this respect, although their amino acid analysis was similar. 5. The proteoglycan fraction isolated from the low-ionic-strength extract shows marked differences in composition from the fractions isolated from the high-ionic-strength extracts. Its protein and glucosamine contents were lower whereas its hexuronic acid and galactosamine contents were higher than those of the latter. It also exhibits major differences in its amino acid composition. The glucosamine:galactosamine ratio of the fraction from the low-ionic-strength extract indicates that it may be an almost exclusively chondroitin sulphate-proteoglycan. Its analysis correlates closely with that of a low-molecular-weight proteoglycan isolated from pig laryngeal cartilage by Tsiganos & Muir (1969). 6. The proteoglycan fractions from both the low- and high-ionic-strength extracts migrate as a single band in zone electrophoresis carried out in a sucrose-density gradient at both pH3.0 and pH7.0, although each showed evidence of band widening during the electrophoresis. All the proteoglycan fractions migrated with the same electrophoretic mobility at pH3.0, irrespective of the differences in composition between them. 7. The differences between the proteoglycans from the low- and high-ionic-strength extracts are discussed and the view is advanced that they may be due to association between predominantly chondroitin sulphate-proteoglycans and a keratan sulphate-enriched proteoglycan species.     

Serglycin and secretion in human monocytes

The human monocytic cell line U-937 has been widely used as a model system for human monocytes. The subclone U-937-B has been adapted to serum-free conditions. This particular U-937 clone and its parent clone U-937-1 were used to investigate the role of the proteoglycan serglycin in human monocytes. For this purpose cells were treated with hexyl-β-D-thioxyloside to abrogate proteoglycan expression. U-937-B cells expressed and secreted exclusively chondroitin sulphate proteoglycans, and after treatment with this xyloside they only expressed and released free chondroitin sulphate chains. Western blotting showed that serglycin core protein was present in conditioned medium of control cells, but absent in medium from xyloside-treated cells. Also, serglycin core protein could be detected in the cell fractions of control cells, but not in the cell fractions from xyloside-treated cells. Furthermore, less proteoglycan-associated proteins could be detected in medium from cells incubated with xyloside, suggesting that the absence of secreted sergycin affects the secretion of such proteins. Cells incubated in the presence of xyloside were analyzed by transmission electron microscopy and shown to contain numerous large empty vesicles. The lack of serglycin, the dominant proteoglycan in U-937 monocyte-like cells, consequently, leads to effects on vesicle formation and secretion of some low molecular weight proteins, suggesting that this particular proteoglycan is of importance for secretory processes in human monocytes.     

Isolation and Purification of Chondroitin 6-Sulfate Protein From Umbilical Cord

Intact chondroitin 6-sulfate protein can be extracted from umbilical cord with dilute saline. Hyaluronic acid which is also extracted, is removed by precipitation with cetylpyridinium chloride followed by washing of the precipitate with aqueous sodium chloride. Subsequent purification is effected by passage through cation and anion exchange resins. Elution from the latter with salt solutions of increasing concentration yields chondroitin 6-sulfate proteoglycan in two fractions. The product is isolated from each of the fractions as the calcium salt by fractional precipitation with ethanol. The protein moiety can be cleaved from the mucopolysaccharide either by proteolytic digestion or treatment with alkali. The results obtaired on reaction with alkali and with sodium borohydride indicate that the polysaccharide is covalently linked to the protein through a serine unit.     

Extracellular glycoconjugates produced by cyanobacterium Wollea saccata

In order to survive in a highly competitive environment, freshwater or marine phototrophic microorganisms have to develop defense strategies that result in a tremendous diversity of compounds from different metabolic pathways. Recent trends in drug research from natural sources have shown that algae and cyanobacteria are promising organisms to furnish novel biochemically active compounds. In this study, we have analysed the extracellular mucilaginous proteoglycan produced by fresh-water heterocytous filamentous cyanobacterium Wollea saccata, strain Hindák 2000/18. This mucilaginous material is an acidic proteoglycan containing 30% protein and 52% carbohydrates on the basis of fraction dry weight. The constituent sugars of the carbohydrate component include glucose, fucose, 3-O-methylfucose, xylose, galactose, 3-O-methylgalactose, mannose, rhamnose, arabinose and glucuronic acid. The extracellular proteoglycan has been separated into five fractions (WF1-WF5) by anion exchange chromatography. Individual polymeric fractions varied in protein (16-57%) and carbohydrate (31-66%) contents, and in the composition of constituent monosaccharides.     

Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.     

Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Protein phosphorylation in the photosynthetic bacterium Rhodospirillum rubrum

Endogenous protein phosphorylation in cellular fractions from Rhodospirillum rubrum was manifested after exposure to [γ-³²P]ATP. At least six phosphorylated protein bands of 90, 86, 64, 31, 13 and 11 kDa were found in the cell-free extract. Treatment of the 64-kDa band with V₈ protease yielded smaller radioactive bands. Phosphoserine, phosphothreonine and phosphotyrosine were detected after acid hydrolysis of the phosphorylated fractions. Protein phosphorylation in all the fractions was insensitive to cAMP, did not recognize exogenous protein substrates and was rapidly reverted upon elimination of the excess of [γ-³²P]ATP. The chlorophyll-anthena apoprotein from R. rubrum chromatophores overlapped the 13-kDa phosphorylated band during gel filtration by high-pressure liquid chromatography suggesting that it is one of the substrates of the protein kinase(s) of R. rubrum.