Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

U50488H、哌唑嗪及心得安对去甲肾上腺素诱导心肌肥大的作用比较

目的：观测κ阿片受体激动对去甲肾上腺素诱导心肌肥大的抑制作用,并与哌唑嗪、心得安作用进行比较。方法：结晶紫染色法测心肌细胞增殖程度;Lowry法测心肌细胞蛋白含量;计算机图象分析系统测心肌细胞体积;[3H]-亮氨酸掺入法测心肌细胞蛋白合成。结果：①低血清环境下,NE明显诱导心肌细胞蛋白含量、蛋白合成及体积的增加,但对增殖无影响。②哌唑嗪和心得安单独作用部分抑制NE诱导的心肌肥大;联合作用则完全抑制。③U50488H明显抑制NE诱导的心肌肥大;其抑制程度与哌唑嗪和心得安联合作用类似,明显高于二者单独作用。结论：NE通过激动α1-和β-肾上腺受体途径诱导心肌肥大。κ阿片受体激动显著抑制NE诱导的心肌肥大,这可能与干预α1-AR和β-AR途径有关。     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Absence of covalently linked core protein from newly synthesized hyaluronate.

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Macromolecules released into the culture medium during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardii.

The culture medium of growing Chlamydomonas reinhardii cells contains hydroxyproline-rich glycoproteins, which are mainly liberated during release of the zoospores from the mother-cell wall. Pulse-labelling studies with [3H]proline and [35S]methionine have been performed in order to detect the protein components released by synchronously growing cells at different stages of the cell cycle. When either [3H]proline or [35S]methionine were applied during the phase of cell growth, radioactive label appeared in the released macromolecules after a lag period of 40 min, whereas incorporation into the insoluble part of the cell wall was delayed only by 20 min. When applied at the end of the growth phase, e.g. 13 h after beginning of the illumination period, the radioactive amino acids were incorporated into the cell wall, but radioactive labelling of macromolecules released into the culture medium could not be detected before the zoospores were liberated from the mother-cell wall. Maximal incorporation of [3H]proline and [35S]methionine into the insoluble part of the cell wall was observed during cell division, but essentially no radioactively-labelled macromolecules were released into the culture medium during this time period. Analysis of the macromolecules, which were liberated during cell enlargement, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed distinct radioactive bands, which were differentially labelled with [3H]proline and [35S]methionine. Among the macromolecules released into the culture medium during cell growth, a component of an apparent Mr 35 000 was preferentially labelled with [3H]proline. This component was also detected after labelling with [35S]methionine, but components of an apparently higher Mr were more prominent after labelling with [35S]methionine. Macromolecules released during the cell-enlargement period of synchronously growing cultures in the presence of [3H]proline contained radioactively-labelled hydroxyproline in addition to proline. These results show that, during cell-wall growth, specific protein components are released into the culture medium and that at least one of these components contains large amounts of proline and hydroxyproline. At least some of these macromolecules seem to be constituents of the cell wall, because during pulse-chase experiments radioactively-labelled macromolecules appeared in the culture medium mainly during the time period when the specific radioactivity of the insoluble inner-cell-wall layer decreased.     

Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation

Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E-115, a murine neuroblastoma cell line. At a concentration of 0.35 microgram/ml, tunicamycin inhibited macromolecular incorporation of [3H]mannose by 75-80%, whereas incorporation of [3H]leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter-binding assay using [3H]quinuclidinyl benzilate ([3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol-induced down-regulation. The rate of mAChR degradation (control t1/2 12-14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [3H]QNB (a membrane-permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [3H]N-methylscopolamine ([3H]NMS, a membrane-impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane.     

Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate-derived product. Recently, it has been shown that free geranylgeraniol (GG-OH) and farnesol (F-OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG-OH and F-OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post-translational modification of key regulatory proteins. In this study we report that GG-OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [³H]thymidine incorporation. The increase in cell number and [³H]thymidine incorporation were significantly lower when F-OH was added. Under these conditions [³H]mevalonate and [³H]GG-OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP-binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Analysis of the proteins metabolically labeled by [³H]mevalonate and [³H]GG-OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG-OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG-OH was added to lovastatin-treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.     

Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function.

The 78,000-dalton glucose-regulated protein (GRP78) and the immunoglobulin heavy-chain-binding protein (BiP) were shown to be the same protein by NH2-terminal sequence comparison. Immunoprecipitation of GRP78-BiP induced by glucose starvation and a temperature-sensitive mutation in a hamster fibroblast cell line demonstrated the association of GRP78-BiP with other cellular proteins. In both fibroblasts and lymphoid cells, GRP78-BiP was found to label with 32Pi and [3H]adenosine. Phosphoamino acid analysis demonstrated that GRP78-BiP is phosphorylated on serine and threonine residues. Conditions which induce increased production of GRP78-BiP resulted in decreased incorporation of 32Pi and [3H]adenosine into GRP78-BiP. Furthermore, we report here that the phosphorylated form of BiP resides in the endoplasmic reticulum and that BiP which is associated with heavy chains is not phosphorylated or labeled with [3H]adenosine, whereas free BiP is. This suggests that posttranslational modifications may be important in regulating the synthesis and binding of BiP.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with [14C]thymidine and [3H]leucine. The evaluation of the DNA turnover and additional [3H]thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. In parallel to the increased rate constant of protein degradation, the cytoplasmic volume fraction of early autophagic vacuoles (AVs) as determined by electron microscopic morphometry was found to be increased twofold in nongrowing 3T3 cell monolayers when compared with the volume fraction of early AVs in growing 3T3 cell monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

3H]Leucine incorporation method as a tool to measure secondary production by periphytic bacteria associated to the roots of floating aquatic macrophyte

The present study assessed the application of [(3)H]Leucine incorporation into protein by periphytic bacteria associated with the roots of the floating aquatic macrophyte Eichornia crassipes. Basic assumptions underlying the method, such as linearity of leucine incorporation, saturation level of incorporation rates, incorporation into other macromolecules, specificity of incorporation for bacterial assemblages and [(3)H]Leucine degradation during samples storage were tested, and two procedures for extracting the incorporated leucine were compared. Both methods gave the same results, however, the hot TCA extraction method was less time consuming than the alkaline extraction method. Incorporation of [(3)H]Leucine was linear for up to 40 min. Saturation concentration of [(3)H]Leucine incorporation into protein was 1500 nM. An experiment with prokaryotic and eukaryotic inhibitors showed no significant [(3)H]Leucine incorporation into eukaryotes even in high leucine concentrations. No significant amounts of radiolabel were incorporated into other macromolecules. The maximum time of sample storage after the incubation is 15 days. The leucine incorporation method can be a reliable tool to measure bacterial production in the periphyton root-associated bacteria.     

Comparative Study of 125I- and [3H]Acetate-Labeled Antibodies in Detecting Iridescent Viruses

Radioimmunoassays for detecting cell-associated or released virus are described using either (125)I- or [(3)H]acetate-labeled antibodies. In the first assay system, antigen-antibody complexes were separated from free antibody by centrifugation. Sensitivities of 0.1 mug of iridescent virus could be achieved with either (125)I- or [(3)H]acetate-labeled antibody. In the second assay, the antigen was fixed to cover-slip cell cultures, and then reacted with labeled antibody, unbound radioactivity being removed by repeated washing. Nonspecific binding with this method was 0.5 to 1% of the total radioactivity added and sensitivities of 0.1 or 10 mug were achieved with (125)I and [(3)H]acetate, respectively. Immunoglobulins were labeled at the rate of 1 in 300 for (125)I and 1 in 200 with [(3)H]acetate although there was a 400-fold greater isotopic abundance of (125)I relative to (3)H. The possibility of preparing labeled protein of high specific activity using carrier-free [2-(3)H]iodoacetic acid is discussed.     

3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters

Vascular endothelial cells previously have been shown to possess a prominent Na-K-Cl cotransport system which mediates a K+ influx of approximately 20 mumols/g of protein/min. Endothelial cell cotransport has also been shown to be regulated by a variety of vasoactive agents and their second messengers, suggesting that the transport system may have an important role in endothelial cell function. In the present study we investigated the possibility that the high level of cotransport in these cells is due to a large number of Na-K-Cl cotransporters in the plasma membrane. This was done by evaluating specific saturable binding of [3H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal [3H]bumetanide binding of 0.83 pmol/mg protein with a dissociation constant of 0.13 microM. From these data, the number of [3H]bumetanide binding sites/endothelial cell was determined to be approximately 230,000, and the turnover number for cotransport activity was calculated to be 300 K+ ions/site/s. These findings indicate that endothelial cells do indeed exhibit a large number of Na-K-Cl cotransporters/cell relative to other cell types. We also investigated the effects on [3H]bumetanide binding of agents known to modulate Na-K-Cl cotransport activity. Saturable binding of [3H]bumetanide was found to be reduced significantly by treatment of the cells with 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol esters, norepinephrine, or rat atriopeptin III, all of which have been shown to inhibit Na-K-Cl cotransport-mediated K+ influx.     

Yeast alpha-factor and somatostatin enhance binding of [3H]estradiol to proteins in rat pancreas and Saccharomyces cerevisiae

Pancreatic tissue contains an [3H]estradiol-binding protein that requires a coligand in the steroid-binding reaction. The endogenous coligand appears to be the tetradecapeptide somatostatin. Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of [3H]estradiol to partially purified pancreatic protein. Supernatant fractions from yeast cells also contain an [3H]estradiol-binding protein. alpha-Factor can enhance specific binding of [3H]estradiol to such yeast fractions. Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of [3H]estradiol but did not inhibit cell growth or induce morphological changes in S. cerevisiae. Thus, it appears that coligand-requiring [3H]estradiol-binding activity and mating in yeast are not directly related.     

12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 82 kDa protein in HL-60 cells

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL-60 and HL-60 Blast II. The latter is a variant which does not differentiate after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). The acylation profiles of the two cell lines as examined by SDS-PAGE differed. TPA induced the myristylation of an approximately 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA-sensitive and TPA-resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)