Incorporation Of 35S-methionine Research Articles

Protein synthesis in bupivacaine (marcaine)-treated, regenerating skeletal muscle.

Skeletal muscle regeneration has been induced by injection of the myotoxic drug bupivacaine (Marcaine) into the rat tibialis anterior muscle. Doses of 1.5 and 1.0% wt/vol produce significant levels of muscle regeneration, but these doses also produce large regions of ischemic muscle. Doses of 0.75 and 0.5% bupivacaine are also effective in inducing regeneration and produce little or no ischemia. Regenerating muscle is significantly more active in the incorporation of 35S-methionine into protein than is control muscle, and the activity increase is directly proportional to the bupivacaine dose injected. Polyribosomes were isolated in greater yield from bupivacaine-treated muscles, as compared with control muscles, 5 days postinjection, and were also more active in cell-free protein synthesis than control polysomes. Again, the yield and activity of the muscle polysomes was directly proportional to the bupivacaine concentration used for injection. Polyacrylamide gel electrophoresis of polysomal cell-free reaction mixtures demonstrated the synthesis of a number of myofibrillar proteins.

The complexity of mitochondrial translation products in mammalian cells

The complexity of mitochondrial translation products in mouse liver and Ehrlich ascites tumor cells have been studied using a mitoplast system active in 35S methionine incorporation. Electrophoretic analysis on gradient polyacrylamide-SDS gels and urea-SDS gels under highly dissociating conditions show that both of the mitochondrial systems synthesize about 22 polypeptides. Many of these 35S labeled products compare with the polypeptides predicted by the DNA sequence analysis data reported by Anderson et al. (1).

Cultured human syncytiotrophoblasts synthesize pregnancy-specific beta-1-glycoprotein (SP1).

The synthesis of pregnancy-specific beta-1-glycoprotein (SP1) was studied in human syncytiotrophoblast cultures from first trimester placentas. Incorporation of 35S-methionine into SP1 was demonstrated by immunoprecipitation and polyacrylamide gel electrophoresis. In double diffusion the precipitation line of neosynthesized SP1 gave a reaction of complete immunological identity with maternal serum SP1. By indirect immunofluorescence intracellular SP1 was localised in the perinuclear region of the cells. The average rate of SP1 release into culture fluid was 600 ng/mg protein in 24 hours. On a molar basis the release of SP1 was 10 times less than that of human chorionic gonadotrophin (hCG). Our results demonstrate, for the first time, that cultured human syncytiotrophoblasts synthesis SP1 immunologically indistinguishable from placental SP1.

Rates of incorporation of radioactive molecules during the cell cycle.

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (3H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) 3H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing 3H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of 3H-TdR and 35S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3H-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of 35S-methionine increased continuously with cell size and DNA content. Incorporation of 3H-TdR in CHO cells was proportional to DNA synthesis.

Hormonal regulation of synthesis of yolk proteins and a larval serum protein (LSP2) in Drosophila.

Juvenile hormone (JH) and 20-hydroxyecdysone (20-HE) have a wide range of roles in insects. They interact during development to determine the quality of the moult1–4 and both hormones have been implicated in control of yolk protein synthesis and ovarian development. However, their functions vary in different insects5–7. In adult Drosophila, they have specific effects on the haemolymph proteins. Both JH analogue (JHA) and 20-HE stimulate the synthesis and secretion of three yolk polypeptides (YPs) into the haemolymph of females8,9. However, whereas JHA stimulates synthesis of YPs in both the fat body and the ovary, 20-HE stimulates the synthesis only in the fat body10, and JH, but not 20-HE, induces YP deposition in oocytes9,11,12. 20-HE, but not JHA, evokes YP synthesis in Drosophila males (which do not normally produce these proteins13), and 20-HE treatment of isolated female abdomens stimulates incorporation of 35S-methionine into a protein of molecular weight 79,000 (designated 79K). We have now compared the responses of the YPs and 79K to combinations of JHA and 20-HE given to both male and female Drosophila and show that 79K is a larval serum protein (LSP2) normally secreted by the fat body of third instar larvae. JHA and 20-HE have additive effects on YP synthesis, but JHA antagonizes 20-HE stimulation of LSP2 synthesis. This effect is probably caused by JHA induction of fat body cell death.

Effects of α-amanitin on programming of mouse blastocyst development

We have investigated the effect of α-amanitin treatment of mouse blastocysts on their further development. Twenty-four-hour treatments with α-amanitin at 1 μg/ml do not interfere with embryo survival or gross morphological development over the next few days although the rate of synthesis of polyA-containing RNA is dramatically reduced during exposure to the antimetabolite. Effects upon rates of protein synthesis are not observed initially, but incorporation of ( 35S)methionine into acid-insoluble material is ultimately inhibited by approximately 50% in all the treated samples. Developmental markers do not all respond in the same way to the α-amanitin treatments used. Whereas levels of β-glucuronidase and plasminogen activator are virtually unaffected by the antimetabolite, α-amanitin treatment interferes with Δ 5,3 β-hydroxysteroid dehydrogenase activity in trophoblast cells and formation of a bilayered inner cell mass. The effects upon the latter markers differ quantitatively with the various exposure periods to α-amanitin. On the basis of these observations, we propose that mRNAs for at least some developmental markers in mouse blastocysts are synthesized long before they are translated.

Cellular synthesis and modification of murine hepatitis virus polypeptides.

Mouse L fibroblasts infected with mouse hepatitis virus, MHV3, and radiolabelled with 35S-methionine, contained, in addition to the three major structural polypeptides, p24, p56 and p180, two additional ones, p22 and p50. Of these total five polypeptides, only three, p22, p56 and p180, were labelled in infected cells during a 2 min 35S-methionine pulse and are, therefore, presumed to be immediate translation products. Pulse-chases and chymotryptic peptide mapping experiments showed apparent precursor-product relationships between p56 and p50 and between p22 and p24. Protein synthesis in infected cells was synchronized at the initiation stage by pre-exposure to hypertonic medium. Using a 0.5 min pulse-10 min chase sequence, to limit incorporation of 35S-methionine to stretches of approx. 100 amino acids adjacent to translational initiation sites, it was found that all three polypeptides, p22, and p56 and p180 contained radiolabel. It is thus apparent that translation of the three major structural proteins (or precursors) is initiated independently rather than at a single site as in the cases of other positive-strand RNA viruses such as polio or Semliki Forest virus.

In vitro translation of cricket paralysis virus RNA.

Cricket paralysis virus RNA acted as a messenger in a translation system and directed incorporation of 35S-methionine into protein. Polyacrylamide gel analysis of the proteins demonstrated the presence of proteins of comparable molecular weight to the viral structural proteins and also potential high molecular weight precursors.

Antigen and Polypeptide Synthesis by Temperature-sensitive Mutants of Respiratory Syncytial Virus

A revised nomenclature for the polypeptides of respiratory syncytial (RS) virus has been devised on the basis of comparison of the Long, A2 and RSN-2 strains by slab-gel electrophoresis. Seven polypeptides, now designated VP200, VGP48, VPN41, VPP32, VPM27, VP25 and VP10, were observed in preparations of all three strains of RS virus, irrespective of the host cell of origin. In addition, a slowly migrating glycopolypeptide GP1 was prominent in partially purified RS virus of the Long and A2 strains obtained from Hep-2 cells, and to a lesser extent from BS-C-1 cells. In the case of the RSN-2 strain, this polypeptide was only resolved clearly in virus obtained from Hep-2 cells. GP1 was an atypical glycopolypeptide in that 35S-methionine incorporation was poor relative to 3H-glucosamine incorporation. The ts mutants of RS virus exhibited four distinct phenotypes with respect to intracellular polypeptide synthesis and antigen production of 39 degrees C. Mutants ts 17 (complementation group B') and ts 19 (group E) were almost completely restricted, suggesting defective early functions. Mutants ts A1 (group A), ts A7 (group C) and ts 1 (group D) synthesized antigen and polypeptides normally, but the amount of antigen at the cell surface was reduced, suggesting maturation defects. In addition, the VPP32 of ts 1 (group D) exhibited an aberrant mobility, confirming its viral specificity. The remaining mutants, representing groups B, F and G exhibited generally impaired synthesis at 39 degrees C. Absence of surface filaments in ts mutant-infected cells at 39 degrees C confirmed their virus-specific nature.

Origins and genetic nonvariability of the proteins which diffuse from maize pollen.

The major function of pollen is to deliver the sperm nuclei to the embryo sac. It does this by germinating and producing a pollen tube and thus provides a relatively simple developmental system for study. Mutants for many pollen functions are accessible, as it is a haploid cell. Mature pollen was fractionated into diffusible proteins, soluble proteins, and proteins insolubly associated with membrane or wall; these protein fractions have been quantified and cataloged by native and SDS polyacrylamide gel electrophoresis. Diffusible proteins are localized in the pollen grain wall whereas soluble proteins are cytoplasmic. The roles of haploid and diploid genomes in specifying these proteins is discussed. Pollen from maximally divergent maize lines was examined for quantitative and qualitative variation in the diffusible proteins. A surprising conservation was found for these proteins indicating some functional role which is, at present, unknown. Initial experiments on the incorporation of 35S-methionine into germinating pollen indicate that major representatives of the diffusible proteins are made within the pollen grain itself. They are presumably included in the pollen wall during development and diffuse out through the pore region. Studies with pollen mRNA and experiments on incorporation of 35S-methionine into developing anthers are underway and will identify the origin of these proteins. A knowledge of the basic developmental biology of maize pollen is a prerequisite to its judicious use as a monitor of environmental mutagens.

Effect of testosterone propionate on protein synthesis by two-dimensional electrophoresis in rat ventral prostate.

Two-dimensional polyacrylamide gel electrophoresis has been used to map the pattern of protein synthesis in rat ventral prostate from both castrate and testosterone treated animals. Proteins were detected by fluorography of 35S-methionine incorporation. In the soluble or cytosol fraction, four major proteins that have molecular weights less than 25,000 and isoelectric points (pI) less then 6.0 and one than has a molecular weight above 30,000 and pI 6.6 are under androgen control. One of these proteins with molecular weight 12,000 and pI 5.4 also appears in the microsomal fraction. Using nonequilibrium pH gradient electrophoresis, soluble proteins of pI greater than 7.0 were investigated. One major protein of 20-25,000 daltons and pI 8.0 appears to be androgen dependent. The ventral prostate from castrate synthesized proteins which are not detectable in androgen stimulated castrates.

Association of polyoma T antigen and DNA with the nuclear matrix from lytically infected 3T6 cells

Nuclear matrix prepared from mouse 3T6 cells lytically infected with polyoma virus retained significant amounts of the 100K T antigen and intact viral genomes. Bound T antigen was resistant to the extraction by high salt (2 M NaCI), detergent (1% Triton X-100) and exhaustive DNAase treatment. Only conditions sufficient to disrupt the integrity of the matrix itself solubilized the matrix T antigen. During the time period of 16–30 hr after infection, both the accumulation (in μg) and the incorporation of 35S-methionine into T antigen increased steadily in cell extracts to a peak at 26 hr and then declined. In contrast, the amount of labeled T antigen retained by the matrix was relatively constant over the same time period. Matrix-bound T antigen was more highly phosphorylated and newly synthesized compared with the extractable T antigen. Viral DNA steadily accumulates in nuclei and on the matrix from 18 to 30 hr after infection. The fraction of viral DNA retained by the matrix was greatest early in infection (25% at 16 hr), declining to less than 10% by 24 hr. These data are consistent with the existence of a fixed (and limited) number of sites for T antigen (more highly phosphorylated) on the matrix and implicate the nuclear matrix as a site of viral DNA replication and possibly encapsidation.

Protein synthesis and degradation during expression of the temperature-sensitive defect in ts A1S9 mouse L-cells.

The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.

Timing of ubiquitin synthesis and conjugation into protein A24 during the HeLa cell cycle

To determine the relationship between formation of protein A24 and replication of chromatin, incorporation of 35S methionine into ubiquitin, protein A24, histone 4 and high mobility group proteins (HMG) 1 and 2 were compared to DNA synthesis and cell division in HeLa cells synchronized by thymidine block. The biosynthesis of ubiquitin and its coupling into protein A24 occurred in parrallel and were largely interphase events rising to a maximum rate in early G1 phase and declining to a minimum during cell division. The ratio of labeling of protein A24 to histone 4 indicated that a high rate of ubiquitin-histone 2A conjugation occurred, in excess of that accounted for by histone synthesis. This additional conjugation was highest in early and late G1 phase and its distribution during the cell cycle was similar to the biosynthesis of HMG 1 and 2.

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF 7, ZR 75 and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of 35S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF 7 cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.

Expression of differentiation antigens in subpopulations of mouse thymocytes: Regulation at the level of de novo synthesis

Mouse thymocytes can be divided into two broad classes by a variety of criteria, including steroid sensitivity, buoyant density, and availability of binding sites for peanut agglutinin. The major class is poorly reactive in immune functional assays, whereas the minor population does contain cells that are immunologically competent. Correlated with these functional differences, the populations differ in their displays of specific surface antigens and in their steady-state terminal deoxynucleotidyl transferase (TdT) activity. To investigate the basis for these phenotypic differences, the two populations were enriched by density gradient fractionation or by differential PNA agglutination; alternatively, the minor population was freed of the major one by in vivo cortisone treatment. These partially purified cells were then examined for their rates of de novo synthesis of four differentiation marker proteins. Incorporation of 35S-methionine into H-2 (K and/or D), TL, TdT, and a Qa-region product (probably Qa-1) was monitored by immune precipitation with specific antisera followed by gel electrophoresis. The metabolic labeling interval was restricted to 30–40 min, to reflect as closely as possible instantaneous rates of synthesis. The two thymocyte populations were found to differ significantly in their production of each of these molecules. Relative to its total protein synthesis, the major population synthesized 10–20 fold less H-2, 5–10 fold more TdT, and 10–20 fold more TL than the minor population. The Qa product was synthesized in both populations, but at a slightly higher rate in the minor population. The biosynthetic rates are consistent with the reported steady-state phenotypes of the cells, and imply that changes in differentiation antigen expression during T lymphocyte development are controlled, at least in part, at a translational or pretranslational level.

Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells

Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by 35S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with 3H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.

De novo protein synthesis by human chondrosarcoma in cell and organ culture: evidence of unusually high collagen production by a neoplastic tissue.

Protein synthesis by human chondrosarcoma tissue and normal articular cartilage was studied in organ and primary monolayer cell culture systems. Protein synthesis by cell cultures was evaluated at 2.7 and 21 days after plating. When compared to normal, incorporation of 3H-proline and 35S-methionine into proteins was elevated in chondrosarcoma samples under both culture conditions. Hydroxyproline analyses of tissue hydrolysates indicated that chondrosarcoma samples contained significantly less collagen than normal articular cartilage, yet were incorporating significantly greater amounts of 3H-proline into 3H-hydroxyproline. Collagen production by cell cultures was assessed by digestion of samples with purified clostridial collagenase. Chondrosarcoma cells produced more collagenase-sensitive protein than normal cells at all intervals. SDS polyacrylamide gel analyses of all preparations showed two collagenase-sensitive proteins with apparent molecular weights of 165,000 and 175,000. Decreased synthesis of another major protein, apparent molecular weight 210-220,000 was noted in chondrosarcoma preparations in both culture systems.

The synthesis and distribution of desmin and vimentin during myogenesis in vitro

Electrophoretic and autoradiographic analyses of the incorporation of 35S-methionine into newly synthesized proteins during myogenesis reveal that presumptive chicken myoblasts synthesize primarily one intermediate filament protein: vimentin. Desmin synthesis is initiated at the onset of fusion. Synthesis rates of both filament subunits increase during the first three days in culture, relative to the total protein synthesis rate. The observed increase in the rate of desmin synthesis (at least 10 fold) is significantly greater than that observed for vimentin, and is responsible for a net increase in the cellular desmin content relative to vimentin. Both filament subunits continue to be synthesized through at least 20 days in culture. Immunofluorescent staining using desmin- and vimentin-specific antisera supports the conclusion that desmin is synthesized only in fusing or multinucleate cells. These results indicate that the synthesis of the two filament subunits is not coordinately regulated during myogenesis. The distributions of desmin and vimentin in multinucleate chicken myotubes are indistinguishable, as determined by double immunofluorescence techniques. In early myotubes, both proteins are found in an intricate network of free cytoplasmic filaments. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, both filament proteins become associated with the Z lines of newly assembled myofibrils, with a corresponding decrease in the number of cytoplasmic filaments. This transition corresponds to the time when the a-actinin-containing Z lines become aligned laterally. These data suggest that the two intermediate filament systems, desmin and vimentin, have an important role in the lateral organization and registration of myofibrils and that the synthesis of desmin and assembly of desmin-containing intermediate filaments during myogenesis is directly related to these functions. These results also indicate that the Z disc is assembled in at least two distinct steps during myogenesis.

On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol.

We have found that tryptose phosphate broth (TPB) prevents the inhibitory effect of chloramphenicol (CAM) on the cell proliferation of chick embryo fibroblasts. Study of growth parameters indicated that no lag or adaptation period appeared necessary for TPB-exposed chick cell populations to grow in the presence of CAM suggesting that a particular cell type was not selected. TPB did not prevent the inhibitory effect of CAM on the mitochondrial protein-synthesizing system. This was supported by cytochrome oxidase activity measurements, studies on the incorporation of 35S-metionine into mitochondrial proteins, electron microscopic observation of alterations in mitochondrial structure. Oxygen consumption was reduced by 95% and cyanide, 2-4-dinitrophenol, and salicylhydroxamic acid do not significantly affect the residual respiration. Analyses of reduced-minus-oxidized-cytochrome spectra of CAM-treated chick cells demonstrate the disappearance of the absorption bands of cytochromes aa3, b559, c1, and c. The presence of a type b cytochrome with maxima at 552 and 557 nm was observed. The results obtained indicate that long-term cultures of CAM-treated chick embryo cells cultivated in the presence of TPB grow with mitochondria devoid of a functional respiratory chain.