Incorporation Of 14C-labeled Amino Acids Research Articles

Transcortin in rat kidney: subcellular distribution of transcortin-synthesizing polyribosomes

The [ 3H]corticosterone-transcortin complexes from kidney cytosol show elution positions on DEAE-cellulose identical to serum transcortin. The incorporation of 14C-labeled amino acids into anti-transcortin-precipitable material of kidney slices has been measured and compared with that of serum transcortin. It was established that kidney synthesized transcortin with an apparent molecular weight of 66 kDa on SDS-electrophoresis which resembles serum corticosteroid-binding globulin. Studies on the binding of [ 125I]anti-transcortin-IgG to membrane-bound rat kidney polyribosomes revealed an association of [ 125I]anti-transcortin-IgG with a discrete polyribosome fraction in the heavy polyribosome region; free polyribosomes were devoid of antigenic material able to bind antibodies to transcortin.

The Short-Term Effects of Inorganic Phosphate on the Levels of Metabolites in Suspension-Cultured Catharanthus roseus Cells

Abstract In order to analyze the effects of inorganic phosphate (Pi) on the energy metabolism of suspen­sion-cultured cells of Catharanthus roseus, short-term changes in levels of glycolytic metabolites were monitored after the addition of 1.25 mм Pi to cultures of cells previously cultured in a Pi-free Murashige-Skoog medium for 24 h. The levels of all phosphorylated intermediates of glycolysis examined were found to increase after addition of Pi. The most striking increases were observed in levels of glucose-6-phosphate and fructose-6-phosphate which rose to approximately 8 (after 30 min) and 15 times (after 120 min) their initial values. The levels of ATP and pyrophosphate doubled during the first 30 min of incubation. In contrast, the level of pyruvate decreased significantly during the initial 30 min and then began to increase again for the next 90 min. The short­ term effects of Pi on the rates of synthesis of proteins and RNA were estimated from the rates of incorporation of 14C-labelled amino acids and uridine into the macromolecules. No appreciable stimulation of the synthesis of proteins and RNA was induced by Pi within 120 min after the addition of Pi. Possible mechanisms are discussed that may be involved in the changes in metabo­lism initiated by the addition of Pi to the cultures.

Transcortin biosynthesis and intracellular distribution of rat liver polyribosomes synthesizing transcortin

Transcortin biosynthesis in rat has been examined using liver slices technique. The incorporation of 14C-labeled amino acids into the anti-transcortin-precipitable material of liver slices has been measured and compared with that of serum transcortin. It was shown that liver synthesized transcortin with an apparent mol. wt of 66 kDa on SDS-electrophoresis which co-migrated with authentic rat serum transcortin. In order to determine an intracellular distribution of transcortin synthesizing polyribosomes, the binding character of [ 125J]anti-transcortin-IgG to free and membrane-bound rat liver polyribosomes has been studied. It was shown that after incubation of [ 125J]anti-transcortin-IgG with liver membrane-bound polyribosomes, the radioactivity was associated with the discrete polyribosome fraction in heavy polyribosome region. In similar experiments the radioactivity of [ 125J]antitranscortin-IgG bound to the free polyribosomes was distributed throughout the polyribosome region. The results of our experiments obviously demonstrated that serum transcortin was synthesized exclusively on membrane-bound polyribosomes of rat liver; free polyribosomes were devoid of detectable antigenic material able to bind antibodies to transcortin.

Inhibition of protein synthesis in mice by ochratoxin A and its prevention by phenylalanine

The sensitivity of protein synthesis to ip doses of ochratoxin A ranging from 1 to 15 mg/kg body weight has been determined in the livers, kidneys and spleens of mice. The incorporation of 14C-labelled amino acids into protein was measured in the total tissue homogenate, in the 105,000-g supernatant fraction and in the fraction of thermostable soluble proteins. The inhibition of protein synthesis was greatest in spleen and kidney and least in liver. The percentage inhibition of protein synthesis by any one dose of ochratoxin A was similar in all of the protein fractions from any one organ. The degree of inhibition of protein synthesis 5 hr after administration of 1 mg ochratoxin A/kg was 26% in liver, 68% in kidney and 75% in spleen. Phenylalanine (100 mg/kg) injected together with ochratoxin A prevented the inhibition of protein synthesis by a dose of 10 mg OTA/kg in all of these organs.

Effect of dietary eggs on liver and plasma proteins of rats. I. Synthesis of serum low-density lipoproteins.

The total lipid content in the livers of rats consuming cooked whole eggs as the only source of food for 21 days was elevated to three times that of control animals. The incorporation of 14C-labelled amino acids into serum low-density lipoprotein apoproteins was increased by 52% in these rats, without affecting the incorporation into serum albumin and liver total proteins. The turnover of low-density lipoprotein in these egg-fed rats appears to be very high.

Larval brine shrimp malate dehydrogenase: biosynthesis and temporal pattern related to environmental salinity.

Brine shrimp nauplii challenged with artificial sea water containing 2.5 M NaCl maintain significantly higher levels of cytoplasmic malate dehydrogenase (s-MDH) than larvae incubated in sea water having 0.5 M NaCl. Eight to ten hours after emergence, free-swimming nauplii living in 0.5 M NaCl exhibit a steady decline of s-MDH for 20-40 hours; the decreased is less and stabilizes earlier-in nauplii incubated in 2.5 M NaCl. The 14C-labeled amino acids produced by H 14CO3 fixation were rapidly incorporated into newly formed s-MDH protein as assayed using quantitative rocket immunoelectrophoresis (IEP) with monospecific antiserum prepared against purified brine shrimp s-MDH. Higher rates of enzyme biosynthesis ( greater than 45%) occurred in 2.5 M NaCl together with rapid s-MDH turnover (half-life = 17 hours), accounting for the difference observed in enzyme level between different salt regimes. In contrast, incorporation of 14C-labeled amino acids into total cytoplasmic protein decreased slightly in high salt, suggesting that a preferential synthesis of s-MDH is taking place. Temporal patterns of s-MDH during embryonic development were monitored using both catalytic activity and quantitative IEP assays. Levels of s-MDH seen in encysted gastrulae (0.22 units or 0.57 microgram s-MDH protein/100 embryos) remain relatively constant through the E1 and E2 emergent stages until the exhibited decline observed in the naupliar stage. The results are discussed in relation to the bioenergetics and temporal development of water and electrolyte regulation in nauplii.

Protein synthesis, amino acid uptake, and pools during isoproterenol-induced hypertrophy of the rat heart and tibialis muscle.

Chronic administration of isoproterenol (ISO) produces hypertrophy of the rat heart and tibialis muscle. With doses of 0.1, 0.3, and 0.6 mg/kg, hypertrophy of the heart is significantly by the 3rd day of treatment. Maximum cardiac enlargement attained with doses of 0.3 and 0.6 mg/kg occurs after 21 days and averages 40% above control values. ISO increases tibialis muscle weight by 15%. Incorporation of 14C-labeled amino acids into total heart and tibialis muscle protein is stimulated by ISO. Maximum stimulation occurs 2-3 h after the fifth daily injection of ISO. The stimulation of incorporation is greater during the first few days of treatment and decreases gradually thereafter. A single injection of ISO decreases the total amino acid concentration of the serum, heart and tibialis muscle whereas the rate of amino acid uptake by the heart and tibialis muscle is increased by ISO. The production of hypertrophy of the heart and tibialis muscle in diabetic or castrated animals by ISO suggests that insulin and testosterone are not essential in the mechanism of ISO-induced hypertrophy.

Inhibition of 14C-labeled amino acid incorporation into protein by particles and surface-active agents in human granulocytes

Contact of human polymorphonuclear leukocytes with latex or zymosan particles resulted in inhibition of incorporation of 14C-labeled amino acids into protein in the phagocytosing cells. The surface-active agents, concanavalin A and digitonin, exerted the same inhibitory activity on 14C-labeled amino acid incorporation into protein in human granulocytes. The inhibition of 14C label incorporation was dose dependent and the half-maximal effect per 10 7 cells was found at 0.8 mg of latex (0.79 μm in diameter), 0.25 mg of serum-coated zymosan and 35 μg of digitonin/ml of the incubation medium. Amino acid transport ( 14C-labeled amino acids or amino[ 14C]isobutyrate) was the same in the resting and phagocytosing granulocytes. These results suggest that the incorporation of 14C-labeled amino acids into protein in human granulocytes is in some way connected to alteration in the cell plasma membrane.

Antigens of a Human Breast Carcinoma Cell Line (BT 20). I. Synthesis of Serum Proteins, Membrane-Associated Antigens, and Oncofetal-Associated Antigens<xref ref-type="fn" rid="FN2">2</xref>

The synthesis of various products by a human mammary cell line (BT 20) was studied by incorporation of 14C-labeled amino acids, choline, glucosamine, or galactosamine into nondialyzable materials. These products had molecular weights ranging from less than 12,300 daltons to more than 200,000 daltons. They were analyzed by immunoelectrophoresis and double diffusion in agar. Among the synthesized products, the following proteins were identified: beta2-glycoprotein I, alpha2HS-glycoprotein, alpha2-lipoprotein, actin, beta2-microglobulin, carcinoembryonic antigen, three oncofetal-associated antigens, and various erythrocyte membrane-associated antigens (namely, glycophorin). Synthesis of milk proteins was not detectable. Only the protein moiety of the glycophorin molecule seemed to be synthesized. The beta2-microglobulin was synthesized in an unbound state as well as bound to a glycoprotein whose relationship with the transplantation or tumor antigens must be determined. The three oncofetal-associated antigens were also synthesized in vitro by human fetal tissues and neoplastic and dysplastic human mammary tissues.

Chronic administration of pentagastrin: Effect on gastric mucosal protein synthesis in rats

Groups of adult rats were injected with either saline or pentagastrin (500 μg/kg) for 14 days. The capacity of the gastric mucosal ribosomes to synthesize endogenous and exogenous mRNA-directed protein in a cell-free system was then investigated. Polyribosomal profiles were also analysed on sucrose gradients. The endogenous mRNA-directed incorporation of 14C-labelled amino acids into protein by the gastric mucosal ribosomes from the pentagastrin-injected rats was found to be considerably greater than that of the control. In the presence of exogenous mRNA (poly-U) the polyribosomes as well as the endogenous mRNA-free ribosomes (run-off ribosomes) from the pentagastrin-treated group showed 86% and 136% increment in [ 14C]-phenylalanine incorporation as compared to the corresponding control preparation. The ribosomal ribonuclease activity between the groups was found to be the same. The results of the present investigation indicate that an enhancement in the translational capacity of the ribosomes is in part responsible for stimulation of gastric mucosal protein synthesis after chronic administration of pentagastrin.

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg ( pO 2). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll. Cells which were incubated at 0.5 mmHg ( pO 2) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of 14C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.

Biosynthesis of streptothricin antibiotics. VII. Origin of streptolidine moiety in antibiotics from Streptomyces species.

The effective production of streptothricin antibiotics by the addition of various amino acids (chemistry defined medium C) and the incorporation of 14C-labeled amino acids into streptothricin-C molecule (organic medium E) were studied with a strain of Streptomyces lavendulae OP-2. It was confirmed that proline, alanine, leucine, and arginine stimulated the production of antibiotics, and arginine and glycine promoted the mycelial growth. Of the amino acids tested, arginine was incorporated preferentially into streptothricin-C, but not tartaric acid or streptolidine itself. Approximately a one-half of radioactivity from arginine incorporated into the antibiotic was located in the streptolidine moiety. Furthermore, from the localization of 14C from arginine in the antibiotic molecule from four strains, a different pattern from that described above was observed. Localization of radioactivity in streptolidine moiety of racemomycin-A from S. lavendulae ISP-5069 was lower than that of β-lysine moiety, suggesting that biogenesis of streptolidine in streptothricins from S. lavendulae strains follows two pathways.

Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3-deficient humans.

Human monocytes synthesized the third component of complement (C3) up to 5 wk in vitro. Evidence for net C3 synthesis was based on (a) incorporation of 14C-labeled amino acids into C3 protein, (b) indentity of the allotype of C3 produced in vitro with that of the doner's serum C3, even in the presence of carrier C3 protein of a different allotype; (c) correspondence of electrophoretic mobility, size, and subunit structure of C3 protein produced in vitro with serum C3; (d) inhibition of C3 production with cycloheximide. Monocytes from two unrelated C3-deficient patients were studied under conditions that supported C3 synthesis by normal monocytes. Serum from each of the patients contained less than 1% of the normal C3 concentration, buth their monocytes produced C3 at approximately equal to 25% of the normal rate when studied after 2 wk in vitro. The C3 produced in vitro by monocytes from one of the patients had the molecular weight of normal serum C3 and dissociated appropriately under reducing conditions. Monocytes from C3-deficient patients could not be distinguished from normals on the basis of morphology, rosetting with C3-coated erythrocytes, or rates of C2, and total protein synthesis.

Preliminary characterization of a glycoprotein having Fc receptor properties extracted from a T cell lymphoma (L‐5178‐Y)

After incorporation of 14C-labeled amino acids and/or of [3H]fucose during in vitro culture of the Thy-1.1-bearing, Fc receptor-positive T lymphoma, L-5187-Y, attempts were made to purify the Fc binding structure(s). Following solubilization of a crude membrane pellet using sodium deoxycholate, the 110 000 x g supernatant was filtered on Sephadex G-200, and the fractions containing IgG-binding material were further purified by affinity chromatography (on IgG Sepharose 4B.) Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the final Fc-binding product showed an apparent molecular weight of 110 000 daltons. On subsequent reduction with 2-mercaptoethanol, five bands (mol. wts. 56 000, 36, 000, 25 000, 18 000 and 15 000) were observed, the latter two being probably degradation products. These results are in accord with some of the published data concerning B cell Fc receptors.

Studies on the lymphocytes of sheep. III. Destination of lymph-borne immunoblasts in relation to their tissue of origin.

Lymph-borne immunoblasts were labeled in vitro with 125I[]iodo-deoxy-uridine, washed and returned by intravenous injection to the sheep from which they had been collected. Twenty h later the sheep were killed and the distribution of the immunoblasts was determined by assaying the radioactivity in various organs. Immunoblasts from the efferent lymph of peripheral somatic lymph nodes (PSLN) went mainly to the spleen, lungs and other PSLN, while immunoblasts from intestinal lymph went mainly to the small gut. This ability of intestinal immunoblasts to home to the gut was demonstrated also in the sterile environment of fetuses in utero; apparently the migratory behavior of immunoblasts, like that of small lymphocytes, is not primarily "antigen-driven". A technique was devised for the collection of peripheral (i.e. afferent to the mesenteric node) intestinal lymph which was found to contain 10-20 times the numbers of small lymphocytes that occur in the peripheral lymph from other tissues. Immunoblasts from peripheral intestinal lymph also homed to the gut. The immunoglobulin content of immunoblasts was studied by making detergent extracts of lymph cells, by applying immuno-peroxidase techniques to cell films and by investigating the incorporation of 14C-labeled amino acids into immunoglobulins by immunoblasts in vitro. Immunoblasts from both somatic and intestinal lymph contained and made IgG and IgM, but many intestinal immunoblasts contained and made IgA. It is not known whether this immunoglobulin mediates the extravasation of immunoblasts into the gut. Nonetheless, there is compelling evidence that there are two major migratory pathways for lymphoid cells; one through the gut-associated lymphoid tissue and the other through the somatic-splenic lymphoid tissues.

Synthesis of the first component of complement by primary cultures of human tumors of the colon and urogenital tract and comparable normal tissue

Primary cultures of normal adult colon from a colonic carcinoma, and urinary tract transitional cells from urethra, bladder, ureter, and renal pelvis, and transitional cell carcinomas from the bladder have been shown to synthesize the first component of human complement for periods up to 54 days. Primary cultures of cells derived from adenoma of prostate, normal prostatic tissue, renal cell carcinoma, and fibroblast did not show evidence of synthesis of C l activity. Net synthesis has been demonstrated by incorporation of 14C-labeled amino acids into C l and its subunit Cl s which were purified by affinity chromatography. When tested the synthesis of C l as well as general protein synthesis was inhibitable by cycloheximide. It has not been ascertained whether all the subunits of C l were produced by the same cell.

The Basis of the Alkalophilic Property of a Species of Bacillus

An alkalophilic bacterium belonging to the genus Bacillus was isolated from an indigo ball. The bacterium exhibited a maximum growth rate at pH 10-0 TO 10-5. The incorporation of 14C-labelled amino acids or [14C]uracil, uptake of 14C-labelled alpha-amino isobutyric acid into the bacterium and oxygen consumption of the bacterium with amino acids as substrates were all maximum at pH 9-0 to 10-5. The uptake of [U-14C]glucose into the organism and oxygen consumption with carbohydrates, on the other hand, showed little variation of rate in the pH 8 to 10 region. The oxygen consumption of intact bacteria or protoplasts in culture medium was maximum at pH 10. The membrane of the bacterium oxidized NADH maximally at pH 7-5, and ATPase bound to the membrane exhibited maximum activity at pH 7.L-Lactate, L-alanine and malate dehydrogenases in the soluble fraction exhibited maximum activities at pH 7-4 to 8-4. The alkalophilic property of the bacterium may be due to the behaviour of the membrane towards charged substances admitted into the organisms.

Studies on the growth ofThiobacillus ferrooxidans

Suspensions ofThiobacillus ferrooxidans in Warburg flasks oxidized ferrous iron and coupled14CO2 fixation to the oxidation. The sulphates of zinc, cobalt, copper, nickel or uranium (0.1–1.0 M) depressed the rate of Fe2+ oxidation: nickel and uranyl ions were the most inhibitory. Uranyl, copper and nickel ions inhibited iron-dependent CO2-fixation, the two former producing a marked uncoupling effect on CO2-fixation, similar to that produced by 2:4-dinitrophenol. Molybdate also inhibited iron oxidation. Incorporation of14C-labelled amino acids and glucose was largely dependent on energy from ferrous-iron oxidation and was also strongly inhibited by uranyl sulphate. Kinetic analysis of the inhibition of iron-oxidation by uranium indicated mixed competitive and non-competitive inhibition. Little binding of238U,63Ni or59Fe toT. ferrooxidans was observed and the effects of uranium were concluded to result mainly from loose binding at sites on the cell membrane concerned with iron-oxidation and possibly the transport of other metals. Molybdate probably interfered with sulphate-dependent steps of iron oxidation. Uncoupling of CO2-fixation probably resulted in part from interference with energy metabolism and could depend on transport of uranyl ions through the cell membrane. CO2-fixation by an uraniumtolerant culture (U+) was less sensitive to uranyl-inhibition than that by the wild-type strain (U−), but iron oxidation and CO2-fixation were much more sensitive to uranium when the U− organisms were previously cultured on thiosulphate rather than ferrous iron.

The synthesis of the small subunit of ribulose 1,5-bisphosphate carboxylase in the french bean Phaseolus vulgaris.

1 Ribulose bisphosphate carboxylase is synthesised de novo during the greening process in the leaves of the French bean Phaseolus vulgaris; synthesis of this enzyme represents about 55% of the soluble protein synthesised, as shown by precipitation of 14C-labelled soluble protein with a specific antiserum to the enzyme. 2 A system in vitro for the completion and release of nascent polypeptides from bean leaf cytoplasmic polysomes was developed; the inclusion of high-speed supernatant and pH 5 enzyme from rat liver was necessary to obtain maximum incorporation of 14C-labelled amino acids into protein. 3 An antiserum to the small subunit of ribulose bisphosphate carboxylase precipitated 30% of the 14C-labelled released protein; after dissociation of the precipitate with sodium dodecyl-sulphate, the radioactivity was eluted from Sephadex G-100 in the same volume as authentic small subunit. 4 Cytoplasmic polysomes, active in synthesising the small subunit, were precipitated by the antiserum to the small subunit and also by an antiserum to the large subunit.

Some Physiological and Biochemical Effects of a Coxiella burneti Lipopolysaccharide Preparation on Guinea Pigs

A lipopolysaccharide obtained in a dialyzed phenol extract from the rickettsia Coxiella burneti produced the following effects in guinea pigs after intraperitoneal injection: hyperthermia, loss of body weight, increased liver weight and concomitant lipid infiltration, elevated levels of hepatic and plasma cortisol, increased incorporation of [(3)H]orotic acid into hepatic 28S and 18S ribosomal ribonucleic acid, increased incorporation of (14)C-labeled amino acids into liver and plasma protein, and leukocytosis. Most of these events also occur during infection of guinea pigs with C. burneti, and a causal relationship between the rickettsial lipopolysaccharide and the biochemical changes that occur during Q fever is suggested.