Incorporation Of Radioactive Leucine Research Articles

Effect of inhibitors of proteolytic enzymes on the growth of normal and polyoma transformed BHK cells

This paper reports the effect of tosyl-phenylalanyl-chloromethyl ketone, Pepstatin, Antipain, Leupeptin and Trasylol, all inhibitors of proteolytic enzymes, on the growth and morphology of normal and polyoma transformed baby hamster kidney cells in culture. Above 1 μM, tosyl phenylalanyl chloromethyl ketone inhibited the growth of both normal and transformed cells to the same extent, and also inhibited the incorporation of radioactive leucine into protein. Pepstatin and Antipain also had no differential effect on the growth of normal or transformed cells, while Leupeptin and Trasylol inhibited the growth of normal cells more than transformed cells.

Synthesis and Degradation of Rat Liver Lactate Dehydrogenase M4: HYBRIDIZATION IN THE PURIFICATION OF LACTATE DEHYDROGENASE ISOENZYMES

Abstract For the measurement of lactate dehydrogenase (LDH) isoenzyme biosynthesis a new method is presented which is based on the quantitative hybridization of isoenzyme M4 with isoenzyme H4. After reversible dissociation the formation of active LDH M4 was more rapid and occurred to a greater extent than the formation of LDH H4. The formation of hybrid isoenzymes from the two subunits resulted in an intermediate total activity. In the reaction mixture, 1 mm NADH resulted in a quantitative recovery of all isoenzymes. Rat liver proteins were labeled after a single injection of [3H]leucine or in double labeling experiments after injections of [14C]leucine and [3H]leucine. LDH M4 (LDH5) of the liver homogenate was separated from the other isoenzymes by DEAE-Sephadex chromatography. The LDH M4 fraction was subjected to a freeze-thaw cycle in neutral phosphate buffer containing 1 m NaCl and 1 mm NADH. A second DEAE-Sephadex chromatography was performed and the LDH M4 fraction obtained was hybridized with nonradioactive LDH H4 with the freeze-thaw technique. A third chromatography on DEAE-Sephadex was performed. The hybridized LDH M subunits attached to the column were eluted with a NaCl gradient. The radioactivity eluted was localized to the fractions containing LDH HM3, LDH H2M2, and LDH H3M. The radioactivity per unit of LDH M4 activity in the liver homogenate was calculated. Using this technique, it was demonstrated that cycloheximide, but not actinomycin D, inhibited the incorporation of radioactive leucine into LDH M4. This indicates that the synthesis of lactate dehydrogenase is coded by a relatively stable messenger RNA. The half-life of rat liver LDH M4 was found to be 4.1 days. The degradation constant, kd, was 0.173 day-1. The synthesis rate, ks, was calculated to be 84 molecules·cell-1·s-1.

Incorporation of radioactive leucine and fucose in brain plasma membrane proteins fractionated by polyacrylamide gel electrophoresis

Plasma membranes were prepared from the cortices of 4–6-day-old rats by homogenization and gradient centrifugation. In vivo labelling of plasma membrane proteins and glycoproteins with leucine and fucose, respectively, was characterized by a delayed appearance of radioactivity in plasma membranes as compared to the homogenate. Major proteins of the plasma membranes had apparent molecular weights of 94 000, 72 000 and 57 000 as determined by gel electrophoresis. Two prominent glycoproteins were detected at about 99 000 and 96 000 daltons. The incorporation of [ 3H]leucine and [ 3H]fucose in plasma membranes was most active in gel regions containing the prominent bands. However, the specific radioactivities of the amido black-stained proteins labelled with [ 3H]leucine and separated by gel electrophoresis indicated slow labelling of the major polypeptides. After 2 or 48 h incorporation, their relative specific radioactivities varied no more than 2-fold.

The effect of nerve growth factor (NGF) upon axoplasmic transport in sympathetic neurons of the mouse

To study a possible agent which controls axoplasmic transport, the rates of transport were examined in postganglionic sympathetic neurons in adult mice which had been treated with nerve growth factor (NGF). Rates were measured by observing the movement along the nerve of a front of radioactive material after a prior intraganglionic injection of L-[4,5- 3H]leucine. The faster of two rates observed in female mice increased from a control value of1.22 ± 0.48mm/h (N= 15) to an experimental value of 1.83 ± 0.53mm/h (N= 6; P < 0.005)following the intramuscular injection of a total of 34 μg NGF/g body weight. In contrast, the same treatment caused the slower of the two rates to decrease from a control value of2.32 ± 0.81mm/day (N = 12) to an experimental value of 1.63 ± 0.70mm/day (N= 12; P < 0.05). The rate of slow transport was also examined in male animals. Untreated male mice exhibit a lower rate of slow transport (1.08 ± 0.38mm/day (N= 9) than do untreated females of equal age ( P < 0.001). The result that nerve growth factor increases the rate of the faster phase of transport, and decreases the rate of the slower phase, indicates that this protein exerts a selective effect on transport. To obtain an independent measure of the influence of NGF, the extent of incorporation of L-[4,5- 3H]leucine into tissue proteins of excised stellate ganglia was assessed after 1 h in organ culture. Animals treated with NGF showed increased incorporation of tritiated leucine. Incorporation of radioactive leucine was correlated with the rateof transport in both control animals ( r = 0.61) animals treaetd with NGF ( r = 0.67). Within the framework of normal biological function, NGF may exert a controlling influence on both phases of axoplasmic transport in sympathetic postganglionic neurons.

Pituitary gland polyribosomal function in rats bearing pituitary hormone-secreting tumors

A cell-free system consisting of ribosomes and cytosol fraction, prepared from rat anterior pituitary tissue, was shown to be active in the incorporation of radioactive leucine into protein. The incorporation of radioactive amino acid resembled protein synthesis in its requirement for ribosomes, cytosol fraction, Mg 2+ and an energy source. The incorporation process was inhibited by puromycin. Sucrose density gradient centrifugation of the ribosome preparations revealed the presence of aggregates of ribosomes as well as single ribosomes. Polyribosomes isolated from pituitaries of control rats consistently incorporated more labeled amino acid into protein than did polyribosomes from pituitaries of rats bearing pituitary tumors. Incorporation by either population of polyribosomes was not changed when cytosol from the alternate source was used. Addition of polyuridylic acid caused an increase in the incorporation of [ 3H] phenylalanine by both polyribosome preparations, however, the functional difference between the two preparations was not eliminated. Sucrose density gradient analysis showed no difference in the sedimentation patterns of polyribosome preparations from pituitary glands of normal and tumor-bearing rats. It is concluded that pituitary polyribosomes from rats bearing hormone-secreting pituitary tumors are less efficient at protein synthesis in vitro than are polyribosomes from glands of normal rats. The defect is suggested to be at the ribosomal level, but the mechanism of this alteration in the ribosomal fraction has not been elucidated.

Comparison of Surface Membranes Isolated from Cultured Murine Neuroblastoma Cells in the Differentiated or Undifferentiated State

Abstract Cloned cell lines derived from the murine C1300 neuroblastoma may be grown in suspension (Spinner) culture. Upon transfer to monolayer culture, morphological differentiation (appearance of long processes) may be induced under a variety of conditions. A method for the isolation of surface membranes from both undifferentiated and differentiated cultures has been developed. The yield (60 to 70 %) is very high and few contaminants were observed. The polypeptide composition of the surface membrane fraction from both types of cultures was very similar as determined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Following incorporation of radioactive leucine into the cultures for 60 hours, the distribution of radioactivity among surface membrane proteins was similar in preparations from differentiated and undifferentiated cells. When radioactive glucosamine was used in a similar experiment there was a preferential incorporation of radioactivity into a protein from differentiated cells with an apparent molecular weight of 105,000. Glucosamine was also preferentially incorporated into a soluble protein of the same molecular weight in differentiated cells. The appearance of this protein on the surface membrane was not due to contamination by the soluble protein, since in differentiated cells radioactive fucose was preferentially incorporated into this membrane protein, but not into the corresponding soluble protein. Synthesis of the glycoprotein of molecular weight 105,000 could also be induced by N6,O2'-dibutyryl cyclic 3' : 5'-adenosine monophosphate (Bt2cAMP) under conditions where morphological differentiation was prevented (Spinner culture). Iodination of proteins exposed on the cell surface was catalyzed by lactoperoxidase in the presence of hydrogen peroxide. A protein of molecular weight 78,000 was preferentially exposed on the surface of differentiated cells. This effect always accompanied morphological differentiation and could not be duplicated by Bt2cAMP treatment of suspension culture cells.

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [(3)H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [(3)H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [(3)H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

Inhibition of cerebral protein synthesis by epileptic seizures without motor manifestations.

Repetitive seizures were induced electrically in 109 male CFN rats that were paralyzed and ventilated with oxygen. Compared with the results in paralyzed, oxygen-ventilated controls receiving sham shocks, incorporation of radioactive leucine into cerebral proteins was decreased up to twofold in the cerebral hemispheres, cerebellum, and brain stem of the experimental animals. The extent of inhibition increased with the duration of seizures. The size of the leucine and protein pools was unaffected by seizures, but the specific activity of the cerebral leucine pool was 61 to 64 percent greater in experimental animals. The findings provide a potential explanation of some of the complications that often follow sustained cerebral paroxysmal activity even in the absence of convulsions.

Talc injury: Changes in glucosamine and leucine incorporation into liver and plasma proteins

Injury was induced in mice by sterile subcutaneous injection of talc. The host response to this injury was studied by measuring the incorporation of radioactive leucine and glucosamine into liver and plasma proteins, and the talc granuloma at various intervals between 2 and 528 hr following talc injection. Incorporation into liver proteins showed a marked transient increase at 21–45 hr following injury. Incorporation into plasma proteins indicated a biphasic response: a marked increase incorporation into the PCA-insoluble fraction at 21 hr with a return to normal values at 45 hr, and a similar marked increase into the PCA-soluble fraction at 45 hr with a gradual return towards normal values. This response was dependent upon the amount of talc injected. A biphasic pattern of incorporation was observed in the talc granuloma also, but this may have been related to the local healing processes.

Nuclear proteins in brain of 7-day-old and adult rats.

Abstract— The incorporation of radioactive leucine into proteins of rat brain was considerably higher in the 7‐day‐old than in the adult rat. The greatest difference in the rate of protein synthesis between these two stages of development occurs in the mitochondrial fraction. Among the nuclear proteins the largest variation was in the histones. The only difference in the relative content of nuclear proteins between 7‐day‐old and adult brain was in the acidic deoxyribonucleoproteins which was higher in the younger animal. The electrophoretic profile of these proteins changed during brain development.

Intracellular amino acids and protein synthesis in Hela cells

(1) When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein. (2) Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected. From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.

Comparison of the synthesis of the light and heavy chains of adult skeletal myosin

1. Incorporation of amino acid into light chains and heavy chains of skeletal myosin has been studied by injection of radioactive leucine into adult rabbits. Light and heavy chain fractions were prepared by dissociation of myosin in 5 M guanidine hydrochloride. Three distinct polypeptide chain species were separated by chromatography on DEAE-Sephadex A-25 with molecular weights of 23 000 (light chain 1), 16 000 (light chain 2) and 12 000 (light chain 3) as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis. 2. The specific activities of myosin light and heavy chains 30 min and 4 h after injection were about 20 and 60 %, respectively, of the values at 24 h. 3. Differences in pool sizes of myosin chains were evaluated by comparing the ratio of specific activities of chains at different times after labeling, since polypeptide chains with larger pools would be expected to become labeled more slowly. The ratio of light chains to heavy chains after 30 min was smaller than after 4 and 24 h, suggesting that the pool of uncombined light chains is larger than that of uncombined heavy chains. 4. Following 4- and 24-h labeling periods the ratio of light chain 1 to heavy chain specific activities was about 0.7, light chain 2 to heavy chain 1.1, and light chain 3 to heavy chain 1.8. It is concluded that these differences in radioactive leucine incorporation after longer labeling periods probably reflect actual differences in synthesis of myosin chains.

Effects of Thyrotropin-releasing Hormone and Hydrocortisone on Synthesis and Degradation of Prolactin in a Rat Pituitary Cell Strain

Abstract Synthesis of prolactin was measured in a clonal strain of functional pituitary cells in culture by incubating the cells with [3H]leucine; labeled prolactin was separated from the other labeled proteins by specific immunoprecipitation followed by dissociation with sodium dodecyl sulfate and disc gel electrophoresis. Accumulation of labeled prolactin in the cells was linear for at least 1 hour; labeled prolactin was released into the culture medium after 1 hour. Treatment with 28 nm thyrotropin-releasing hormone for 3 days increased prolactin accumulation in the medium 2.5-fold, and the increased synthesis of prolactin completely accounted for the increased accumulation. Treatment with 5 x 10-6 m hydrocortisone for 7 days decreased prolactin accumulation in the medium 2- to 5-fold, and the decrease in synthesis completely accounted for the decreased accumulation. Therefore the long term (3 to 7 days) effects on these cells of thyrotropinreleasing hormone and hydrocortisone result only in a change in the synthesis of prolactin and not in its rate of degradation. Pulse-chase experiments, using 10 µg per ml of cycloheximide or 10 mm leucine to block further incorporation of [3H]leucine into prolactin, show that there is no detectable degradation of prolactin under the usual conditions of culture. Incubation with 0.1 mm leucine does not prevent incorporation of radioactive leucine into prolactin and results in a 1.7-fold increase in labeled prolactin within 4 hours after the start of the chase. This result suggests there are two intracellular pools of leucine in GH cells, one of which does not appear to equilibrate readily with the medium.

Biosynthesis of growth hormone in the rat anterior pituitary gland. Stimulation of biosynthesis in vitro by insulin.

The effect of insulin on the incorporation of radioactive leucine into growth hormone was investigated by using rat anterior pituitary glands incubated in vitro. A 50% stimulation over control values was observed at insulin concentrations above 2mum (280munits/ml). The effect was specific for growth hormone biosynthesis, over the range 1-5mum-insulin (140-700munits/ml). Lower more physiological concentrations had no significant effect in this system. Above 10mum (1.4 units/ml) total protein synthesis was also increased. The stimulation of growth hormone synthesis could be partially blocked by the addition of actinomycin D, suggesting that RNA synthesis was involved. Insulin was found to stimulate the rate of glucose utilization in a similar way to growth hormone synthesis. 2-Deoxyglucose and phloridzin, which both prevented insulin from stimulating glucose utilization, also prevented the effect of insulin on growth hormone synthesis. If glucose was replaced by fructose in the medium, the effect of insulin on growth hormone synthesis was decreased. We conclude that the rate of utilization of glucose may be an important step in mediating the effect of insulin on growth hormone synthesis.

The distribution of ribosomes between different functional states in livers of fed and starved mice

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with (3)H or (14)C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.

Interrelationship between amino acid pools and protein synthesis in the rat submandibular gland

The uptake and incorporation in vitro of radioactive leucine in rat submandibular gland fragments have been studied. Both uptake and incorporation were found to be influenced by the concentration of leucine in the medium. By means of a cold-wash procedure we were able to distinguish between extra- and intracellular free amino acids. We found that it takes 10 to 15 min for the radioactive leucine to reach maximal concentration in the intracellular soluble amino acid pool. Nevertheless their incorporation into protein becomes linear shortly after the start of incubation in the radioactive medium. We did not find any sign of a decreasing rate of protein synthesis during the incubation. In chase-type experiments, the incorporation of radio-active leucine stops immediately when the labelled medium is replaced by unlabelled medium, though the free, intracellular amino acid radioactivity declines only slowly. These results confirm earlier reports, that in vitro, under conditions of sufficient extracellular amino acid supply, the amino acid molecules incorporated into protein are not selected from the intracellular pool.

Haemoglobin messenger RNA in spleen of anaemic rabbits

Haemoglobin meseenger RNA was isolated from spleen erythroïd cells of anaemic rabbits. The mRNA fraction looks quite homogeneous when analyzed by sucrose gradient centrifugation; it sediments in the 9S region. When added to an ascites cell free system, spleen 9S RNA stimulates the incorporation of radioactive leucine into protein. The synthesized product has been characterized and identified as α and β chains of rabbit globin.

Effects of cycloheximide on the response of intestinal mucosa to cholera enterotoxin.

Prior studies have indicated that effects of cholera enterotoxin (CT) on the small intestine are delayed in onset and involve an interaction with adenyl cyclase in the mucosa. It has also been shown that the administration of cycloheximide to rabbits in doses which inhibit crypt cell mitoses (20 mg/kg), diminishes CT-induced fluid production in jejunal loops. These latter studies have been interpreted as indications that CT-related intestinal secretion is a crypt cell function and that it is mediated by a CT-induced protein. The present study was undertaken to delineate more precisely the nature of the interaction in the intestine between cycloheximide and cholera toxin. Pretreatment of rabbits with cycloheximide reduced by 60% the secretory response to CT in isolated ileal loops with intact blood supply. Sodium and chloride flux measurements on mucosa isolated from these and control loops indicated that this antisecretory effect of cycloheximide persists in vitro. Measurements of radioactive leucine incorporation into mucosal protein indicated that the dose of cycloheximide employed inhibited protein synthesis by 90%. This inhibitory effect was shown to be independent of any effect of cycloheximide on amino acid uptake across the brush border. Measurements of adenyl cyclase activity and cyclic AMP levels in ileal mucosa of cycloheximide pretreated and control animals indicated that cycloheximide did not diminish the CT-induced increases in these parameters. These observations demonstrate that cycloheximide reduces CT-induced intestinal fluid production without interfering with the CT-induced augmentation of adenyl cyclase activity or the consequent rise in cyclic. AMP concentration. Since the antisecretory effect of cycloheximide persists in vitro, it probably involves a direct interaction of the antibiotic with mucosal cell ion transport mechanisms rather than an indirect effect mediated by other humoral or neurogenic factors. The present observations also suggest that the secretory response of the intestine to CT involves neither the synthesis of new adenyl cyclase nor that of a protein modifying its activity.

Role of luminal lecithin in intestinal fat absorption.

AbstractThe effects of biliary lecithin on fat absorption were studied in 1 day bile fistula rats fed micellar solutions of bile salt, monoglyceride and radioactive free fatty acids. By electron microscopy and measurement of uptake of radioactivity into liver and adipose tissue, it was shown that in the absence of bile lecithin there was significant impairment of fat release from mucosa. Fat clearance was effected by the feeding of phosphatidyl choline or choline, but not phosphatidyl ethanolamine, inositol or cholesterol. In the absence of luminal choline there was a decrease in incorporation of radioactive leucine into mucosal protein. It is concluded that biliary and dietary lecithin or choline play an important role in triglyceride transport out of intestinal mucosa, by providing surfactant lecithin for the chylomicron envelope and by supporting mucosal protein biosynthesis.

Studies on the assembly and secretion of a multicomponent protein. The biosynthesis of vitellogenin, an oestrogen-induced glycolipophosphoprotein.

1. Incorporation of [(32)P]P(i) and [(3)H]leucine into vitellogenin secreted in vitro by liver slices from oestrogen-treated Xenopus laevis is accompanied by a 2h lag; no lag is apparent for the incorporation into total tissue protein. 2. The addition of cycloheximide was found immediately to inhibit further incorporation of radioactive leucine into total tissue protein. The incorporation into secreted vitellogenin, however, continued for 2h after the addition of cycloheximide. 3. Pulse-labelling of liver slices with [(3)H]leucine for 30min, followed by a chase with a large excess of unlabelled leucine, resulted in the appearance of radioactivity in secreted vitellogenin from 90min after the end of the pulse period. 4. Evidence is presented which suggests that of the radioactivity from [(3)H]leucine incorporated into proteins by the liver of oestrogen-treated Xenopus some 70% is present in the single protein vitellogenin. 5. The incorporation of [(32)P]P(i) into vitellogenin followed a pattern identical with that found for [(3)H]leucine in the pulse-labelling experiments and this indicates that synthesis of the polypeptide chain and incorporation of P(i) are closely linked processes. 6. The cumulative evidence suggests that the 2h lag phase represents the time required for the assembly and secretion of this multicomponent protein.