The uptake and intracellular distribution of [35S]trithiomolybdate in bovine liver in vivo

[35S] trithiomolybdate was administered intravenously to a group of four steer at two dose rates, 1 and 26 mg Mo per animal. Radioactivity appeared rapidly in the liver and was distributed in all the subcellular fractions examined. Examination by Sephadex G-100 gel-filtration of the cytosol fraction showed that distinct 35S-binding protein peaks were present. The protein-bound radioactivity was displaceable and was identified as [35S]thiomolybdates. No radioactivity eluted with metallothionein, but 35S was associated with the high molecular weight copper fraction, eluted in the void volume of the column, which increased transiently after the administration of the higher dose. It was suggested that the presence of protein-bound thiomolybdates in the liver gave rise to new ligands, which altered the equilibrium of copper between the different metal-binding proteins. This might be similar to the alteration in the copper-binding of albumin produced by the presence of thiomolybdates.     

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.     

Cartilage fucoproteins with sites for cross-linking by transglutaminase

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of gamma-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15,000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.     

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.     

Studies on myelin-basic-protein methylation during mouse brain development.

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.     

Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules

The keratinous capsules surrounding rat sperm mitochondria were isolated 24 days after intratesticular injections of [⁷⁵Se] selenite or [³⁵S] cysteine. Dodecyl sulfate-polyacrylamide gel electrophoresis of purified, doubly labeled mitochondrial capsules revealed only a single ⁷⁵Se-labeled component, whose molecular weight was 17,000, in agreement with previously reported observations obtained with cruder sperm fractions. Most of the ³⁵S label and the major zone of stained protein on the gels coincided with the position of ⁷⁵Se, suggesting that selenium is associated with a cysteine-rich structural protein. The level of selenium in rat sperm, 195 ± 3.2 ng/10⁸ sperm (approximately 30 ppm), determined by hydride generation and atomic absorption spectrophotometry, is consistent with a structural function for this trace element in the sperm.     

Studies on the biosynthesis of sulfolipids in the Diatom Nitzschia alba.

Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.     

Biochemical changes in rat kidney on exposure to elemental mercury vapor: effect on biosynthesis of metallothionein.

Evidence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a metallothionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rats which were injected with [U-14C] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-14C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporation of 109Cd into the fraction followed the same pattern as that of [14C] cystine in rats injected with tracer doses of CdCl2 labeled with radioactive 109Cd isotope. This 10 000 mol. wt. protein, newly synthesized in response to repeated exposures to mercury vapor, exhibited identical properties to metallothionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [U-14C]-cystine into this protein in rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney.     

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.     

Membrane fractions from the outer layers of spores of Bacillus thuringiensis with toxicity to lepidopterous larvae.

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.     

Accumulation of cadmium in rat liver cadmium binding protein following single and repeated cadmium administration.

The accumulation of cadmium in rat liver cadmium binding protein induced by single and repeated intraperitoneal injections of CdCl2 and the de novo biosynthesis of CdBP were studied by using 109Cd to measure cadmium binding in the CdBP and 35S incorporation as indicator of protein synthesis. The biosynthesis of CdBP is controlled by the cadmium concentrations. For single doses up to 1 mg Cd2+/Kg b.w. about 50% of the cadmium is present in the soluble fraction of liver bound to CdBP and the incorporation of 35S-cysteine is linear with the cadmium concentration. When single doses ranging from 1 to 3 mg Cd2+/Kg b.w. are administered the fractions of both 35S-cysteine and cadmium incorporated into de novo synthesized CdBP gradually decrease. For single doses higher than 3 mg Cd2+/Kg b.w. the biosynthesis capability is maximum and 20 mug Cd/g liver can be incorporated into the de novo biosynthesized CdBP. When rats are treated every day with amounts of cadmium of about 0.8 mg Cd2+/Kg b.w. for up to 8 days a dose-proportional increase in both Cd incorporation and CdBP biosynthesis are observed. This shows a cumulative incorporation of cadmium in the de novo biosynthesized CdBP. Experiments carried out by injecting 65ZnCl2 and 203HgCl2 every day showed that they are not accumulated like cadmium and do not induce the biosynthesis of rat liver CdBP after repeated administration over 7 days.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts.

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [gamma-32P]ATP and [gamma-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occurred at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min. The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phosphorylation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular weight of about 100 000 and the ATP-specific main component a molecular weight of 110 000. The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Sulphate utilization in an aphid symbiosis

When two clones of Myzus persicae were maintained on a defined diet with inorganic sulphate as sole sulphur source, their growth and survival were inferior to that on diets containing the sulphur amino acid, methionine. This discrepancy is due, at least in part, to the phagostimulatory properties of methionine, which stimulated aphid feeding rate by 50–150%. Myzus persicae incorporated radioactivity from dietary [³⁵S]sulphate into protein and low molecular weight compounds, including cysteine and methionine. Two lines of evidence indicate that the mycetocyte-symbionts are responsible for the reductive assimilation of sulphate. (1) [³⁵S]sulphate incorporation is abolished by treatment of the aphids with the antibiotic chlortetracycline, which disrupts the symbionts; and (2) [³⁵S]sulphate is utilized by isolated embryos (which contain mycetocyte-symbionts but no gut flora) but not by isolated guts. Tracer studies suggest that 20% of dietary radiosulphur is translocated to the aphid tissues, and it is hypothesized that methionine may be the principal product released by the symbionts.     

Effect of streptozotocin-diabetes on rat liver asialoglycoprotein receptor turnover: in vivo degradation and in vitro biosynthesis

The metabolic turnover of the Hepatic Binding Protein (HBP) was investigated in streptozotocin-diabetic rats. We have already shown that diabetes induced a decreased ligand binding capacity while the immunoreactive HBP was normal. To explore the eventual modifications due to diabetic state upon the turnover of HBP, we followed the in vivo degradation of HBP and its biosynthesis in vitro. After in vivo labelling with L-[3H] leucine and purification of HBP from rat livers, we found a 20% decrease in diabetic HBP half-life. By in vitro incubations of freshly isolated hepatocytes and a 2 h-pulse in the presence of L-[35S] methionine, we showed that diabetes provokes an increased uptake of L-[35S] methionine in hepatocytes allowing an augmented synthesis of HBP although the L-[35S] methionine incorporation into total proteins was less efficient.