AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

THE EFFECT OF MORPHINE ON THE TRANSPORT AND METABOLISM OF INTRACISTERNALLY-INJECTED LEUCINE IN THE RAT

—Intracisternally injected l or d-[¹⁴C]leucine was retained longer in the brains of morphine-treated rats than in saline-injected control animals. This resulted in higher levels of the labelled leucine and of labelled metabolites of the l-isomer in free pools of brain tissue. However, the absolute levels of brain amino acids and the relative distribution of radioactivity among l-leucine metabolites in brain were unaffected by treatment with morphine, indicating that no disturbance of leucine oxidation through the citric acid cycle was produced by the drug. The inhibition of protein synthesis caused by acute administration of morphine was calculated to be greater than previously reported since morphine treatment increased the specific radioactivity of the free pool of leucine in brain following the intracisternal injection of the labelled amino acid. Possible mechanisms responsible for these morphine effects are discussed.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.     

FAST AND SLOW COMPONENTS IN AXONAL TRANSPORT OF PROTEIN

(a) After injection of labeled leucine into the eye of goldfish, radioactive protein rapidly accumulates in the contralateral optic tectum in the layer containing the synaptic endings of the optic fibers. This material reaches the tectum 6–12 hr after the isotope injection, a fact which indicates that the rate of transport is at least 40 mm per day. (b) This rapidly transported material has been shown to consist exclusively of protein, in which the label remains attached to leucine. (c) Inhibition of protein synthesis in the retina prevents the appearance of the transported protein in the tectum, but inhibition of protein synthesis in the tectum does not. Substances having some of the same properties as leucine, such as cycloleucine and norepinephrine, are not transported to the tectum. These experiments all indicate that the transported protein is synthesized in the retina. However, inhibition of retinal protein synthesis after this protein has been formed does not interfere with the transport mechanism itself. (d) The fast component consists of about 85% particulate material. It may be distinguished from a slowly moving component, transported at 0.4 mm per day, which contains about 5 times as much radioactivity as the fast component, and which consists of 60% particulate matter and 40% soluble protein.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

THE INCORPORATION AND FATE OF H3-TYROSINE IN THE HAIR CORTEX OF RATS OBSERVED BY RADIOAUTOGRAPHY

The incorporation of H³-tyrosine into the protein of the cells in the cortex of rat hair has been investigated by radioautography. In growing hairs, radioactivity is found in the matrix, the upper bulb, and the whole of the keratogenous zone up to the fully keratinized part of the shaft, 10 and 30 minutes after an injection of labelled tyrosine. This is unequivocal evidence of protein synthesis at these sites. There is a very precise relationship between the end of protein synthesis and the hardening of the cortical cells at the top of the keratogenous zone. The way in which the silver grains of the radioautographs are clustered indicates that at 30 minutes after the injection the isotope is distributed more evenly in the matrix and upper bulb than in the top of the keratogenous zone. Possibly this reflects a difference, at these sites, in the cell components engaged in protein synthesis, or in the proteins being synthesized. The fully keratinized and hardened part of the hair was not radioactive at 10 and 30 minutes after the injection of H³-tyrosine. The rate at which the radioactivity moves into this region shows that the hair of rats grows 0.9 mm/24 hours. Comparison of the degree of radioactivity along the growing hair in the 30-minute, 12-hour, and 36-hour materials shows conclusively that protein accumulates in the cortical cells during their keratinization. An injection of a labelled amino acid does not behave as an ideal pulse dose; consequently, the grain density over the hair cortex at 36 hours is 100 per cent larger than would be expected if an ideal pulse dose situation existed.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells.

A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.     

RENEWAL OF GLYCEROL IN THE VISUAL CELLS AND PIGMENT EPITHELIUM OF THE FROG RETINA

The renewal of glycerol in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with [2-³H]glycerol. Assay of chloroform:methanol extracts showed that the labeled precursor was used mainly in lipid synthesis, although there was also some utilization in the formation of protein. Radioactive glycerol was initially concentrated in the myoid portion of rods and cones, indicating that this is the site of phospholipid synthesis in visual cells. The glycogen bodies (paraboloids) of accessory cones were also heavily labeled, suggesting the diversion of some glycerol into glycogenic pathways. In the pigment epithelium, only the oil droplets became significantly radioactive. The outer plexiform layer (which contains the visual cell synaptic bodies) and the cone oil droplets gradually accumulated considerable amounts of labeled material. Within 1–4 h, labeled molecules began to appear in the visual cell outer segments, evidently having been transported there from the myoid portion of the inner segment. Most of these were phospholipid molecules which became distributed throughout the outer segments, presumably replacing comparable constituents in existing membranes. In rods only, there was also an aggregation of labeled material at the base of the outer segment due to membrane biogenesis. These highly radioactive membranes, containing labeled molecules of lipid and protein, were subsequently displaced along the rod outer segments due to repeated membrane assembly at the base. The distribution of radioactivity supported the conclusion that membrane renewal by molecular replacement is more rapid for lipid than it is for protein.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

INCORPORATION OF 3H-LABELLED LEUCINE INTO THE PROTEIN FRACTION IN THE RETINA OF THE HONEYBEE DRONE

Abstract— Protein synthesis in the retina of the honey-bee drone was studied by incubating head slices in labelled leucine and measuring the TCA insoluble radioactivity. It was found that the protein-bound radioactivity in illuminated retinas was half of that in dark-adapted ones. This ratio was not affected by pre-treatment with puromycin. It was therefore concluded that, in the drone, the main influence of illumination is to increase the rate of breakdown of proteins.
Relatively high concentrations of labelled protein were found in dark-adapted retinas when the retinula cells were hyperpolarized by bathing the preparation in a sodiumfree medium; low concentrations were found when retinula cells were depolarized by increasing the extracellular potassium concentration. These findings suggest that protein metabolism of the retina is influenced by the membrane potential of retinula cells.     

Toxoplasma gondii: Growth in the absence of host cell protein synthesis

Host cell protein synthesis continues when cultured cells are infected by Toxoplasma gondii. In order to determine if this host function is necessary for the parasite we used two independent methods that specifically block cellular protein synthesis. In the first, we infected a temperature-sensitive Chinese hamster ovary cell mutant that has a thermolabile leucyl tRNA synthetase. At the restrictive temperature of 40 C, the mutant cells showed only negligible protein synthesis that was probably mitochondrial. At this temperature, the growth and nucleic acid synthesis of T. gondii proceeded normally and [³H]leucine was specifically incorporated into the parasite as demonstrated by autoradiography. A secpnd method for blocking protein synthesis by the host cell employed treatment of uninfected human fibroblast cells with muconomycin A, an inhibitor of initiation. Repeated washing of monolayer cultures reduced the free muconomycin A to an insignificant level while the cells remained incapable of protein synthesis. T. gondii infected and grew normally in the inhibited cells. Autoradiographic localization of the incorporation of [³H]leucine showed that it was almost exclusively in the intracellular parasites in the cells pretreated with muconomycin A. In the untreated control most of the [³H]leucine was incorporated by the host cell rather than the parasite. We conclude that de novo protein synthesis by the host cell is not required to support the growth of intracellular T. gondii.     

PROTEIN SYNTHESIS IN THE BRAIN OF OCTOPUS VULGARIS, LAM

Abstract— The process of protein synthesis in the brain of Octopus vulgaris Lam has been examined after systemic administration of [³H]leucine and upon incubation of the tissue in sea water containing the radioactive precursor. After injection of [³H]leucine in the branchial heart, the radioactivity of the TCA-soluble fractions of the three main brain divisions reached a maximum in about 30 min and decreased thereafter, while incorporation into the protein fractions was complete in approx. 2 h. Per unit wet weight the radioactivity of brain proteins was higher than that of most other organs. In vitro the rate of incorporation of [³H]leucine in the protein fraction of the optic lobe remained low for more than 1 h, but increased several fold thereafter. Preincubation of the tissue in sea water abolished the lag period. Similar effects were observed in the vertical lobe as well as in the optic lobe of young and adult octopuses but not in the white body, a non-nervous organ. The process of protein synthesis in the optic lobe is markedly inhibited by puromycin, cycloheximide and chloramphenicol. Electrophoretic analysis on polyacrylamide gels indicated that the soluble proteins labelled in vitro and in vivo are similar.     

THE RELATIONSHIP BETWEEN DNA SYNTHESIS AND NUCLEAR PROTEINS IN THE CEREBRUM AND CEREBELLUM OF 8-DAY-OLD RATS

In-8-day-old rats the higher rate of DNA replication in cerebellum than in cerebrum is accompanied by an enhanced synthesis of nuclear proteins. The greatest difference between the incorporation of tritiated leucine into proteins of cerebral and cerebellar cell nuclei occurs in the acid-soluble deoxyribonucleoproteins. However, the specific radioactivity of the acidic deoxyribonucleoproteins is similar in both tissues. The relative content of these proteins and the activity of the RNA polymerase is higher in cerebrum than in cerebellum. The results suggest that in the cerebrum of young rats these proteins are mainly concerned with the regulation of RNA synthesis.     

The role of phenylalanine in differentiating amphibian melanocytes

To see whether phenylalanine serves as a substrate in melanogenesis, hanging drop explants of neural crest from amphibian (Ambystoma maculatum and A. mexicanum) embryos were subjected on the seventh day in vitro to treatment with phenylalanine-³H and studied by means of light microscopic radioautography. All melanin-containing cells showed label. On the other hand, when puromycin, an inhibitor of protein synthesis, together with the labeled amino acid was administered to the cultures, no radioactivity was incorporated by pigmented cells. Comparable results were obtained when leucine was substituted for phenylalanine. In control experiments, puromycin and labeled tyrosine or 3,4-dihydroxyphenylalanine (DOPA), both known precursors for melanin synthesis, were administered to the neural crest cultures. In these experiments, puromycin had no effect on the incorporation of label by pigmented cells. Our data strongly indicate that in differentiating amphibian melanocytes with functional pigment-forming systems, phenylalanine is used in protein synthesis, but does not serve as a substrate for the tyrosine-tyrosinase system.In another series of experiments, explants of neuroepithelium (neural crest anlage) were grown from the time of explantation to the seventh day in vitro in the presence of phenyllactic acid, an analog of phenylalanine. Pigment cells developed normally.These results suggest that phenylalanine plays little or no role in pigment cell differentiation.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.