Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

Protein-bound ADP in Myogenic and Chondrogenic Cells

These experiments suggest that chondroblasts, cells notoriously sessile, contain considerable amounts of protein (or proteins) that binds ADP in a manner which is indistinguishable from actin. These findings are consistent with the in situ decoration experiments¹ which reported that chondrocytes form arrow-head complexes when treated with HMM. What is unexpected is the relatively large amounts of ADP-binding protein in chondroblasts compared with well developed skeletal myotubes. It is not clear why relatively immotile cells should possess such sizeable quantities of an actin-like molecule subserves functions other than those usually associated with contraction.     

Distribution of Protein-bound Hexosamine in Chloroplasts

Intact chloroplasts of spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) mesophyll cells contained 0.33, 0.50, and 0.14% of bound hexosamine on a protein basis, respectively. Undifferentiated maize chloroplasts contained 0.19%. Values for chloroplast lamellae were, respectively, 0.16, 0.18, 0.12, and 0.06% and for envelope membranes they were 1.6, 2.5, 3.8, and 2.7%. Thus most of the hexosamine of chloroplasts is located in the envelope membrane.     

Lipid components of sialosylgalactosylceramide of human brain

A ganglioside, previously designated HG-B in our laboratory, was isolated from mixed human brain ganglioside preparations and shown to contain equimolar quantities of sialic acid, galactose, and sphingosine. Treatment of this material with neuraminidase yielded a galactosylceramide. The ganglioside, now referred to as sialosylgalactosylceramide, thus appears to be identical with G(gal) reported by Kuhn and Wiegandt. The fatty acids and long-chain bases of this material were analyzed by gas-liquid chromatography. Approximately equal amounts of normal and hydroxy acids were found. Oleic, palmitic, and stearic acids were the only normal fatty acids present. In the hydroxy series, the C(24) and C(23) saturated acids were the major components. The ratio of C(20) to C(18) long-chain base was approximately 5:3. These data suggest that sialosylgalactosylceramide has no direct metabolic relationship with either the major brain gangliosides or adult brain cerebroside.     

Protein-bound oligosaccharides of Semliki Forest virus.

Semliki Forest virus was grown in BHK cells and labeled in vivo with radioactive monosaccharides. Pronase digests of the virus chromatographed on Bio-Gel P6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson, J. and Clamp, J.R. (1971) Biochem. J. 123, 739-745.) The former was labeled with [3H]fucose, [3H]galactose, [3H]mannose and [14C]glucosamine, the latter only with [3H]mannose and [14C]glucosamine. The three envelope glycoproteins E1, E2 and E3 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E1 and E3 revealed glycopeptides of A-type. E2 revealed glycopeptides of B-type. E2 yielded additionally a glycopeptide (Mr3100) which was heavily labeled from [3H]galactose, but only marginally from [14C]glucosamine, [3H]fucose and [3H]mannose. Whether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E1 and 4000 in E3; the B-type unit of E2 had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggest that E1 and E3 contain on the average one A-type unit; E2 probably contains one 3100 dalton unit plus one or two B-type units.     

Protein-bound kynurenine is a photosensitizer of oxidative damage

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.     

Protein-bound phosphorylserine in acid hydrolysates of brain tissue. The determination of [32P]phosphorylserine by ion-exchange chromatography and electrophoresis

1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from (32)P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ;phosphorylserine' fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [(32)P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ;phosphorylserine' fraction by 53+/-11%, as compared with only 19+/-5% for the phosphorylserine isolated by electrophoresis.