Incorporation Of Radioactive Leucine Research Articles

Immunological studies on the incorporation of radioactive leucine into sea urchin embryonic proteins: extractability and turn-over of newly synthesized proteins

This study was undertaken in order to investigate the turn-over of newly synthesized sea urchin embryonic proteins, the distribution of these proteins in the cell and their changes in solubility with time. Refined immunological methods providing unique possibilities for studies of individual proteins were utilized. The proteins were obtained in two soluble fractions: a concentrate of proteins from the cytosol was obtained by homogenization in a hypotonic medium and another fraction constituting membrane-associated proteins was solubilized by detergent treatment of the residual pellet; 75% of the total protein content in early and 60% in later developmental stages was thus solubilized. The proteins solubilized by these mild methods retained their antigenicity and could be analyzed by immunodiffusion methods. The fate of newly synthesized embryonic proteins was studied by incorporation of a radioactive amino acid using two different incubation procedures. The results of these studies indicated that newly synthesized proteins changed their solubility properties with time. Proteins were thus transfered from the soluble to the insoluble fraction. The findings that more proteins became insoluble during development mentioned above also supports such an interpretation. The synthesis of individual proteins was studied by immunodiffusion and autoradiography. Most of the antigens in the two soluble fractions were identical. Experiments with absorbed antisera revealed that only two to four antigens were present exclusively in one of the two soluble fractions. Three groups of antigens differing in regard to their metabolic activities were distinguished. Some antigens were labeled after both procedures without substantially increasing in concentration during development. Thus, their labeling was probably sustained by turn-over. Other antigens were labeled only after one of the two incubation procedures indicating large variations in regard to synthesizing rates of individual proteins.

Synthesis and degradation of folate reductase in sensitive and methotrexate-resistant lines of S-180 cells.

The methotrexate-resistant AT-3000 line of S-180 cells has at least 150-fold more immunologically cross-reactive folate reductase than sensitive cells. Highly specific immunologic and protein purification procedures were used to show that the increased enzyme levels in this line are due to a corresponding increase in the rate of folate reductase synthesis. This observation indicates that the relative turnover of the enzyme is not significantly different in the two lines. Folate reductase was purified to homogeneity from both the sensitive and the methotrexate-resistant cells. Comparison of various physical, kinetic, and immunochemical properties of the enzymes revealed no differences. These observations suggest that the AT-3000 line contains one or more regulatory variations leading to the over-production of folate reductase protein that is similar, if not identical, to that produced by sensitive cells. In resistant cells, specific immunoprecipitation experiments demonstrated that folate reductase comprises as much as 7 to 8% of the continuously labeled soluble protein and 6 to 7% of the soluble protein synthesis. Growth of these lines in the absence of methotrexate resulted in a slow decrease in the level of folate reductase to less than 1%. This decrease corresponded to a similar decrease in the relative rate of enzyme synthesis. Variations in the level of folate reductase with cell growth are also due to changes in the relative rate of enzyme synthesis. In the AT-3000 line, pulse decay experiments showed that the half-life of folate reductase was long (50 hours) relative to cell doubling time (24 hours), and also that methotrexate had little or no effect on the turnover of the enzyme. Comparison of the incorporation of radioactive leucine into folate reductase in continuous and pulse labeling experiments gave independent confirmation of these results. Therefore, the relative rate of folate reductase synthesis was the major parameter determining the amount of folate reductase under all examined conditions that resulted in altered levels of the enzyme in resistant cells.

Effects of Estrogen on the Turnover Rates of Ornithine Aminotransferase in Rat Liver and Kidney

The turnover rates of ornithine aminotransferase in the liver and kidney of control rats and those treated with estrogen were determined by injecting L-[14C]leucine (U) and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzymes. The half-life of the ornithine aminotransferase [EC 2.6.1.13] in the liver (t1/2=0.95 days) was significantly different from that of the kidney enzyme (t1/2=4.0 days). Studies on the incorporation of radioactive leucine into ornithine aminotransferase in the kidney under steady-state conditions showed that the rate of synthesis of this enzyme after treatment with estrogen was 5 to 6 times higher than that in untreated animals. The rate constant of degradation of kidney ornithine aminotransferase under steady-state conditions induced by estrogen treatment was not significantly different from that under control conditions. The results showed that the increase in the rate of biosynthesis, not to a decrease in the rate of degradation. No significant changes in the rates of biosynthesis and degradation of liver ornithine aminotransferase were observed after administration of estrogen.

Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with beta-naphthoflavone or phenobarbital.

The administration of polycyclic aromatic compounds such as beta-naphthoflavone or 3-methylcholanthrene is known to cause the induction of many liver microsomal monoxygenase activities and the appearance of a distinct cytochrome called P-448 in genetically responsive, but not in nonresponsive, inbred mouse strains. However, the administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin induces these activities and cytochrome P-448 formation to the same extent in both responsive and nonresponsive inbred strains. In contrast, phenobarbital or pregnenolone-16 alpha-carbonitrile induces in both responsive and nonresponsive strains a different profile of enzyme activities and the appearance of cytochrome P-450 (rather than cytochrome P-448). In the present studies, electrophoresis of liver microsomal proteins from inbred C57BL/6N and DBA/2N and recombinant inbred AKXL-38 and AKXL-38A mouse strains revealed the presence of four polypeptides whose relative staining intensity could be correlated with the induction state of the microsomes as determined by enzymatic and spectral methods. Of these four bands, Band 4 (55,000 daltons) was increased whenever spectral measurements revealed an increase in the cytochrome P-448 content due to administration of beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Administration of pregnenolone-16alpha-carbonitrile caused an increase in Band 3 (54,000 daltons), whereas administration of phenobarbital caused an increase primarily in Band 2 (51,000 daltons) but also smaller increases in Band 1 (49,000 daltons) and Band 4. The changes observed for phenobarbital and pregnenolone-16alpha-carbonitrile were the same for both responsive and nonresponsive strains. The same electrophoretic technique was used to measure the incorporation of radioactive leucine into microsomal proteins. Microsomes were prepared from liver combined from responsive mice (C57BL/6N) treated with beta-naphthoflavone and L-[14C]leucine and nonresponsive mice (DBA/2N) treated with beta-naphthoflavone and L-[3H-4,5]leucine. A significant increase in the 14C/3H ratio was observed for Band 4, and decreases were seen for Bands 1 and 2. In similar experiments with other mice and phenobarbital as the inducing agent with L-[14C]leucine and the vehicle alone with L-[3H-4,5]leucine, the 14C/3H ratio was markedly increased for Band 2, and smaller increases were observed for Bands 1 and 4. These results and other data presented indicate that the increased formation of cytochrome P-448 and P-450 by beta-naphthoflavone and phenobarbital, respectively, is primarily the result of an increased rate of de novo protein synthesis rather than a decreased degradation rate or a conversion of pre-existing polypeptides.

Inhibition of thyroglobulin biosynthesis and degradation by excess iodide. Synergism with lithium.

Lithium and excess iodide inhibit the release of thyroid hormone from preformed stores. We thus tested the hypothesis that this was due to an inhibition of thyroglobulin breakdown. Rats were pre-treated with propyl-thiouracil (PTU) for 3 weeks in order to deplete their thyroids of thyroglobulin. While the PTU was continued, lithium chloride (0.25 mEq./100 g weight) or potassium iodide (3 mg per rat) were injected every 12 h for d days. Thereafter the thyroglobulin content in thyroid gland homogenates was measured. PTU pre-treatment lowered the thyroglobulin content from 4.21 to 0.22 mg/100 mg gland. Lithium caused a marked re-accumulation of thyroglobulin to 0.60 mg/100 mg within 3 days. While iodide alone had only a borderline effect, it markedly potentiated the action of lithium and a combination of the two drugs increased the thyroglobulin content to 1.04 mg/100 mg. Thyroxine was injected into similarly pre-treated animals to suppress secretion of thyrotrophic hormone. This markedly inhibited the proteolysis of thyroglobulin and 1.3 mg/100 mg gland accumulated after 3 days. Excess iodide, given in addition to thyroxine, decreased the amount of thyroglobulin accumulated to 0.75 mg/100 mg gland. To study whether this could be explained by an inhibitory action of iodide on thyroglobulin biosynthesis, thyroid glands from animals treated with excess iodide were incubated in vitro in the presence of 0.2 mM iodide for 3 h. Iodide decreased the incorporation of radioactive leucine into total thyroidal protein and into thyroglobulin by 25 and 35% respectively. Iodide did not inhibit protein synthesis in the kidney, liver or muscle tissue. Thus, large doses of iodide selectively inhibit thyroglobulin biosynthesis.

Thyroxine-binding globulin biosynthesis in isolated monkey hepatocytes

Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyoxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6 h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium had increased to 300%, indicating probable thyroxine-binding globulin secretion.

Cytoplasmic synthesis of soluble and mitochondrial malate dehydrogenase isozymes in maize

The effects of cycloheximide and chloramphenicol on the incorporation of radioactive leucine into trichloroacetic acid-insoluble material and on malate dehydrogenase (MDH) activity in maize scutella were studied. In 40 h of treatment, chloramphenicol (0.5 – 2.0 mg/ml) does not inhibit the increase of either soluble (s) or mitochondrial (m) malate dehydrogenase isozymes. However, 8 h following the addition of cycloheximide (2–10 μg/ml), the usual increase of total malate dehydrogenase activity is reduced by more than 70%. The reduction in the activity of the soluble and the mitochondrial malate dehydrogenase isozymes is similar. From these observations, and from our former studies on this system, we conclude that both the soluble and the mitochondrial malate dehydrogenases are synthesized on cytoplasmic ribosomes.

Steroid-induced early protein synthesis in rat uterus and prostate.

An early 'induced protein', after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double-labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21-day-old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labelled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labelling in vitro with radioactive acetate. Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol-treated vs non-stimulated uteri being corrected for variations of the acid-soluble radioactive leucine pool. Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful. The contribution of both negative and positive findings in steroid-induced early protein synthesis is discussed in the context of the current knowledge of hormone action.

Chromosomal proteins of rat brain: increased synthesis and affinity for DNA following a pulse of the carcinogen ethyinitrosourea in vivo.

A single pulse of ethlnitrosourea (EtNU), administered to 10-day-old BD IX-rats, specifically results in a high incidence of neuroectodermal tumors in the central and peripheral nervous system. At five days after an EtNU-pulse, analyses of protein-DNA interactions were performed using chromatin dissociation and re-association experiments, following incorporation of radioactive leucine into brain chromosomal proteins (CP) during short-term suspension culture. In comparison with 15-day-old control animals, the brain cells of EtNU-treated rats exhibited (i) an increased rate of CP synthesis, and (ii) an increased affinity of the newly-synthesized CP for brain DNA of both control and EtNU-treated animals.

Polyadenylic acid-containing RNA from rat brain synaptosomes.

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)‐cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)‐cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one‐half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)‐containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA‐precipitable material in a cell‐free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.

Ethanol inhibition of reticulocyte protein synthesis: the role of haem.

Ethanol, in concentrations of 0.05-0.8 M, inhibited intact human and rabbit reticulocyte protein synthesis in the presence of iron-transferrin for endogenous haem synthesis. Associated with this effect there was a conversion of polyribosomes to monoribosomes and a decreased incorporation of radioactive leucine into nascent globin chains. When physiological levels of ethanol (0.05-0.1 M) were used, these effects were prevented by incubation with 50 muM haemin and reversed by removing the alcohol and reincubating with iron-transferrin or haemin. The polyribosomal disaggregation was also prevented by stopping ribosomal movement with 5 mM cycloheximide. Neither ATP nor GSH levels were altered in the presence of ethanol. When non-physiological levels of 0.8 M ethanol were used, haemin did not prevent the inhibition of protein synthesis. Likewise, in the rabbit reticulocyte cell-free lysate system containing haemin inhibition was noted at concentrations greater than 0.05 M ethanol. The polyribosomal disaggregation in reticulocytes incubated with 0.8 M ethanol was associated with decreased dissociation of monoribosomes into subunits. Similarly, when ribosomes were directly suspended cell-free in 0.1 or 0.8 M ethanol there was a decreased percentage of subunits. These results indicate that physiological concentrations of ethanol inhibit initiation of reticulocyte protein synthesis secondary to a block in haem synthesis. When intact cells are exposed to high non-physiological concentrations of ethanol the inhibition is secondary to decreased ribosomal dissociation. The cell-free lysate inhibition is also through this effect on ribosomal dissociation. This study supports the view that alcohol is a direct toxin to developing red cell precursors via its effect on mitochondrial haem synthesis. The physiological role of the decreased dissociation of monoribosomes into subunits is not yet clear.

Malaria studiesin vitro

Aotus trivirgatus monkeys with established infections of Plasmodium falciparum were treated orally with either chloroquine or the novel compound 1-amidino-3-(3-chloro-4-cyanophenyl) urea. Blood samples were cultured in vitro, 18 hours after treatment, when no morphological abnormalities were apparent. The incorporation of radioactive leucine from the medium by the blood of treated monkeys was compared with that of the undosed control. Parasite maturation was also examined. Both chloroquine and the amidinourea were effective against the drug sensitive strain of P. falciparum. The combined use of an in vivo and in vitro test demonstrated that biochemical disturbance of the parasite may be demonstrable before morphological effects are seen. This system should prove useful in drug metabolism studies and in experiments using large or expensive animals.

Immunoglobulin turnover in B lymphocyte subpopulations.

"In vitro" turnover of leucine-labeled and of radioiodinated IgM has been studied with cells from various lymphoid organs of nude mice, i.e. lymph nodes, thoracic duct, spleen and bone marrow, as well as with subpopulations of B cells from spleen and bone marrow separated by free flow electrophoresis. Three types of IgM-producing lymphocytes could be distinguished by their turnover rates of IgM, by the size of the released IgM and by the capacity of the IgM molecules to be labeled by the lactoperoxidase-catalyzed radioiodination reaction and/or by incorporation of radioactive leucine. Type I cells release 7-8 S IgM rapidly (t1/2 = 1-3h); the released IGM is leucine-labeled and radioiodinated. Type II cells release 7-8 S IgM slowly (t1/2 =10-30); the released IgM is leucine labeled and radioiodinated. Type III cells release 19 S IgM rapidly (t1/2 =2-4 h); the released IgM is leucine labeled, but not radioiodinated. Lymph nodes and thoracic duct contain predominantly type II cells, bone marrow contains type I and II cells, spleen contains type I,II and III cells. It is suggested that type III cells are Ig-secreting plaque-forming plasma cells, type II cells are small, resting "memory" B cells, and type I cells may be newly formed antigen-inexperienced B cells.

Influence of cycloheximide on the lung

We examined the time course of the influence of cycloheximide on descending pressure-volume curves of excised lungs and on protein and lecithin synthesis and oxygen consumption by lung slices. We also looked at the influence of cycloheximide on granular pneumocyte ultrastructure. Excised lungs from cycloheximide-treated animals are more compliant than controls. After ventilation with air, lungs from control and cycloheximide animals show increased retractive forces and a shift to the right of the deflation P-V curve. Incubation at 38 degrees C for 30 min reverses these changes in control lungs, but not in lungs from cycloheximide-treated rabbits. There is no change in liquid delfation P-V curves after cycloheximide. Cycloheximide causes an immediate decrease of 50% in incorporation of radioactive leucine into protein by lung slices. Incorporation of radioactive palmitate into lecithin and oxygen consumption are also decreased by 50% 6 h after cycloheximide. Lamellar bodies in granular pneumocytes are smaller after cycloheximide. Cycloheximide causes a significant increase in the surface density of the lamellar body envelope. Cytoplasmic area of granular pneumocytes is increased after cycloheximide.

Activation of myosin synthesis in fusing and mononucleated myoblasts

The synthesis of the heavy chain subunit of myosin has been studied in breast muscle myoblasts from embryos of the Japanese quail, Coturnix coturnix japonica, during differentiation of these cells in culture. Specifically, these experiments were done to examine the roles of myoblast fusion and the regulation of myoblast cell division in the control of myosin heavy chain synthesis. The rates of myosin heavy chain synthesis have been quantitated in cultures of fusing myoblasts by measurement of the incorporation of radioactive leucine and valine precursors into myosin heavy chain, and simultaneous determination of the intracellular specific activities of these radioactive amino acids. These measurements demonstrate that, prior to fusion, dividing myoblasts synthesize little, if any, myosin heavy chain, but that during the period of myoblast fusion, myosin heavy chain synthesis becomes activated at least 50 to 100-fold. Myosin heavy chain synthesis was also measured in mononucleated myoblasts inhibited from fusing by the presence of EGTA in the culture medium. These experiments demonstrate that myosin synthesis can be activated in mononucleated myoblasts to reach rates similar to those attained in fused myoblasts. This activation occurs under conditions in which EGTA-inhibited myoblasts were induced to withdraw from the cell division cycle by reducing the concentrations of the serum and embryo extract components of the culture medium or by prior “conditioning” of standard growth medium. These experiments, therefore, establish that the activation of myosin synthesis in breast muscle myoblasts does not require fusion, but indicate that activation is co-ordinated with the withdrawal of myoblasts from the cell division cycle.

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.

Protein Synthesis in Mouse Brain During Development of Acute Morphine Tolerance

In order to observe the effects of morphine on protein metabolism in mouse brain, experimental procedures were carried out over a 7 hr period of infusion. When the analgesia reached a peak, namely around 2 hr after the start of infusion, the in vivo incorporation of radioactive leucine into protein estimated by the dual label technique was uniformally depressed in all the examined subcellular fractions of both brain and liver. After tolerance developed, however, the incorporation of leucine increased to a much higher level than the control in brain subcellular fractions and the increase was masked by naloxone. In contrast, the incorporation into a TCA soluble fraction of the brain S2, separated by Gray and Whittaker’s method, was more than doubled even after 4 hr infusion. While the in vitro incorporation rate of the mitochondrial fraction significantly fluctuated during development of tolerance with naloxone that of the synaptosomal fraction did not fluctuate. The observed coincidence in the time course of development of tolerance and changes in the brain protein synthesis indicates a possible relationship between the phenomena, though the causal nature of the relationship could not be elucidated.

Inhibition of Synthesis of Peroxidases and Some Proteins by Heliangine inPhaseolus mungoHypocotyl Sections

The peroxidase activity in Phaseolus mungo hypocotyl sections floated on water decreased during the first 3 h and then increased again. The activity of this enzyme was reduced by heliangine and cyeloheximide in vivo, but not in vitro. Peroxidase activity is inhibited by heliangine and cycloheximide since the enzyme is not synthesized. Disk gel electrophoresis studies revealed that at least six species of proteins, soluble in Tris-HCl, pH 7.6, were synthesized in Phaseolus hypocotyl sections during 24 h incubation on water. Heliangine inhibited the synthesis of two of them, a species of peroxidase and another protein. Heliangine inhibited the incorporation of radioactive leucine into the cell wall fraction, suggesting that it inhibits synthesis of cell wall proteins. It did not, however, inhibit the incorporation of labelled proline into the cell wall fraction. The results suggest that heliangine inhibits the synthesis of only some proteins.

Proceedings: Control of prolactin biosynthesis in vitro by insulin.

Prolactin synthesis and release can be investigated in vitro by incubation of the anterior pituitary gland. When isolated rat anterior pituitaries are incubated in vitro with labelled amino acids, radioactivity is incorporated into prolactin, growth hormone and other pituitary proteins. The anterior pituitary has been established as an insulin-sensitive tissue (Goodner & Freinkel, 1961) and Goodner & Krouse (1972) reported that insulin stimulates protein synthesis in the rat anterior pituitary gland. Insulin is required for a response of mammary-gland explants to prolactin and these two hormones act together in the regulation of alveolar cell differentiation during pregnancy. Betteridge &Wallis (1973) showed that insulin stimulates the biosynthesis of growth hormone in oitro. It was therefore decided to investigate the effect of insulin on prolactin synthesis in a system in vitro. Anterior pituitary glands were obtained from Sprague-Dawley rats and incubated in Krebs-Ringer bicarbonate medium containing 1 1.2m~-glucose and rH]leucine (5pCi/ ml). Paired hemi-pituitaries were used for experimental and control observations. Incubations lasted for 8h at 37°C in a humified C02+ air (5:95) atmosphere. At the end of this period the glands were removed from the medium, weighed and homogenized. Portions of the media and pituitary homogenates were precipitated with trichloroacetic acid and analysed for incorporation of radioactive leucine into total pituitary protein. Prolactin and growth hormone were separated as discrete bands by polyacrylamidegel electrophoresis of further portions of media and pituitary homogenates. The prolactin content was determined by densitometry of the stained polyacrylamide gels. Incorporation of radioactive leucine into prolactin was estimated by scintillation counting after excising the appropriate band and solubilizing with HzO2. Total prolactin synthesis was calculated by combining results from media and pituitary homogenates. In this system in oitro prolactin is actively synthesized and released. Over the 8 h incubation period, the rate of prolactin synthesis and release increases fairly linearly, as does the rate of total protein synthesis. Pituitary glands from mature female rats synthesized more prolactin than those from male rats. Total prolactin synthesis, expressed as a percentage of the total pituitary protein synthesis, was 20% in females and 3 % in males. Control of prolactin synthesis and release was further investigated by using female rats. The effect of bovine insulin on the biosynthesis of prolactin in the female anterior pituitary gland was investigated with this system in oitro. Over the range 1 m to 1~

Protein biosynthesis by the pulmonary alveolar macrophage: conditions of assay and the effects of cigarette smoke extracts.

The quantitative and qualitative patterns of protein synthesis by the pulmonary alveolar macrophage were investigated by measuring the uptake of radioactive leucine into protein. A preliminary analysis comparing noninduced and bacille Calmette Guerin-induced cells (BCG cells) indicated that the 2 groups of cells have similar qualitative and quantitative protein synthetic patterns, although the BCG cells were able to synthesize protein for longer periods of time. The ability of pulmonary alveolar macrophage to incorporate radioactive leucine into protein in the presence and absence of aqueous smoke extracts was then studied in detail. Smoke extracts affected the time course and the extent of incorporation of radioactive leucine into protein, causing an inhibition of incorporation after approximately 15 min of incubation. The inhibitory effect was directly proportional to the amount of smoke extract and was at least partially reversible. Cells treated with smoke extracts released a greater percentage of the...