The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.     

Characterization of over-expressed alkaline phosphodiesterase I in tumour-derived fibroblasts from patients with neurofibromatosis

Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6·0. The enzyme required Zn²⁺ for its activity, was heat labile, and nicked superhelical covalently closed circular ?X174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430 000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.     

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphorylation state, glycosylation state, copy number, isoelectric point, and molecular weight.     

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.     

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.     

An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo

Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by [35S]methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 (or about 5%) of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg.     

Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells

Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with [32P]orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.     

Purification and properties of the clotting enzyme from limulus lysate

The clotting enzyme from Limulus lysate which is involved in the gelation reaction of lysate with endotoxin has been purified and some of its properties determined. It was isolated from endotoxin-treated lysate and purified by gel filtration, ion exchange chromatography, and disc gel electrophoresis. Reaction of clotting enzyme with lysate clottable protein produces a clot or gel such as occurs with the gelation of lysate by endotoxin. Purified clotting enzyme has an approximate molecular weight of 84,000 (subunit MW 43,000), is isoelectric at pH ca. 5.5, trypsin-like, heat labile and pH sensitive.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases.     

Purification and characterization of a neutrophil chemotactic factor from Dirofilaria immitis

A neutrophil chemotactic factor (NCF-Di) was purified from a crude extract of Dirofilaria immitis adult worm by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. NCF-Di showed a single protein band by both polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. The molecular weight of NCF-Di was estimated to be 17,000 by gel filtration on Sephadex G-150, and 14,000 by SDS-PAGE. NCF-Di was an acidic protein with isoelectric point of 4.5. NCF-Di was absorbed neither to lentil lectin-Sepharose nor to concanavalin A-Sepharose. The chemotactic activity of NCF-Di was heat labile (56 C, 1 hr), but was resistant to periodate oxidation. These results suggest that NCF-Di is a simple peptide which has few or no sugar chains. These physicochemical properties of NCF-Di were compared to previously reported parasite-derived chemoattractants or purified allergen of D. immitis.     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.     

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.     

L(+)-Lactate binding to preparations of rat hepatocyte plasma membranes

Incubation of rat hepatocyte plasma membranes with L-[14C]lactate resulted in the labeling of protein(s) of apparent molecular weight 40,000 when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The binding was saturable, irreversible, and inhibited by pyruvate, 2-oxoglutarate, and alpha-cyano-3-hydroxycinnamate, but not by D-lactate. It was markedly enhanced by L-alanine, but not D-alanine or beta-alanine. The binding protein(s) could be solubilized in cholic acid giving a single peak on gel filtration corresponding to a molecular weight of 26,000 and an isoelectric point of 5.1. This peak, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ran in a position corresponding to an apparent molecular weight of 40,000. When membranes were treated with Triton X-100, lactate binding was retained by the Triton-insoluble fraction. The binding of L-[14C]lactate increased with incubation time, due apparently to the appearance of new binding sites and not to sequestration into vesicles. As many of the characteristics of lactate binding to rat hepatocyte plasma membranes were found to be similar to those of lactate entry into isolated hepatocytes, we speculate that the lactate-binding protein could represent part or whole of a plasma-membrane lactate transporter. Lactate-binding proteins of the same molecular weight were identified in the plasma membranes from rat erythrocytes, cardiac muscle, skeletal muscle, lung, and brain.     

Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of l-[(35)S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (gag) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with d-[(3)H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the gag proteins and that several of these polyproteins are glycosylated. A comparison of gag precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.     

Characterisation of ISAV proteins from cell culture.

The characterisation of 2 infectious salmon anemia virus (ISAV) proteins is described. Proteins were harvested from ISAV-infected Chinook salmon embryo (CHSE)-214 cell culture by continuous elution denaturing gel electrophoresis, enabling the harvest of specific molecular weight fractions. Through the use of a polyclonal antiserum to ISAV, it was possible to identify a potentially autolytic major antigen of 72 kDa and a glycosylated protein of approximately 38 kDa which varied in size depending on cell line compatibility. N-terminal amino acid sequencing of the glycosylated proteins suggests that it is encoded by segment 6 of the ISAV genome. Further, sequence analysis of the glycosylated protein account for the variable molecular weight and may explain differences in host cell compatibility.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

The identification of a normal rat gene located close to the gene for the potential myoepithelial cell calcium-binding protein, p9Ka

The potential calcium-binding protein p9Ka is related to S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. p9Ka accumulates abundantly in cultured rat mammary myoepithelial-like cells but is very much less abundant in the parental cuboidal epithelial cells. p9Ka mRNA is found in normal rat mammary gland, and preliminary experiments suggest that it is found in the mammary myoepithelial cells. A 17-kilobase pair fragment of cloned normal rat DNA contains the gene for p9Ka, but it also contains the gene for two additional polypeptides of molecular mass 6 kDa that are resolved as two isoelectric focusing variants by two-dimensional gel electrophoresis. These two isoelectric focusing variants correspond to two abundant polypeptides present in the cultured myoepithelial cells and probably arise from postsynthetic modification of the product of a single gene. The mRNA for the product of this gene and the p9Ka mRNA are both found in the normal rat mammary gland, but these two mRNAs are differentially expressed in certain tumor-derived rat cell lines.