Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth,haemolymph components and uric acid excretion in the silkworm, Bombyx mori

Effects of various kinds of dietary protein on growth of the silkworm, Bombyx mori, were determined using semi-synthetic diets. Also, the ingestion, digestion and utilization of dry matter and of nitrogen were measured. Nutritive effects of dietary proteins and supplementation of limiting amino acids on haemolymph protein and amino acids pattern were also investigated. Larval growth was largely dependent on the dietary proteins. When the larvae were reared on a diet containing weakly nutritive proteins such as gluten and zein, haemolymph protein was decreased and uric acid excretion was markedly accelerated. The free amino acid composition of the haemolymph manifested characteristic patterns according to the kinds of dietary protein.The supplementation of gluten and zein with their limiting amino acids resulted in a rise of haemolymph protein and a drop in uric acid excretion. The amino acid patterns in the haemolymph were greatly changed according to supplementation.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Uric acid in nucleic and amino acid synthesis in the boll weevil, Anthonomus grandis

When uric acid-2-¹⁴C was injected into the boll weevil, Anthonomus grandis, it was metabolized to RNA, DNA, amino acid, and ¹⁴CO₂ at the end of 2 hr. The free amino acids, lipoamino acids, and protein amino acids were all labelled with the free amino acids showing the highest specific activity. Incorporation in DNA was slight, but it was extensive in RNA; cytidylic acid showed the greatest amount of incorporation.     

The incorporation of glycerol and lysine into the lipid fraction of Staphylococcus aureus

1. Incubation of washed cells of Staphylococcus aureus with [1-¹⁴C]glycerol results in the incorporation of glycerol into the lipid fraction of the cells. The rate of incorporation is increased by the presence of glucose and amino acids. The presence of amino acids increases incorporation into the fraction containing O-amino acid esters of phosphatidylglycerol. 2. Glycerol, incorporated into washed cells by incubation with glycerol, glucose and amino acids, is rapidly released from the lipid fraction when cells are incubated at low suspension densities in buffer. 3. Of nine amino acids tested, only lysine is significantly incorporated into the lipid fraction. The incorporation is increased by the presence of glycerol, glucose and other amino acids, especially aspartate and glutamate. 4. The incorporation of lysine is increased by the addition of puromycin at concentrations that inhibit protein synthesis. Chloramphenicol does not increase the incorporation of lysine but abolishes the enhancing effect of puromycin. 5. The enhancing effect of puromycin is accompanied by a similar increase in the incorporation of lysine into the fraction soluble in hot trichloroacetic acid. 6. Lysine is incorporated into the lipid fraction that contains O-amino acid esters of phosphatidylglycerol and corresponds in properties to phosphatidylglyceryl-lysine. 7. Lysine is rapidly released from the lipid of cells incubated in buffer only at low suspension densities. 8. Incubation of cells with the phosphatidylglyceryl-lysine fraction does not lead to the appearance of free lysine or to incorporation into the fraction insoluble in hot trichloroacetic acid.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

Fate of [14C]-glycine injected into ligated Galleria mellonella L. larvae: Formation of purines and uric acid riboside

After injection of [U¹⁴C]-glycine into the haemolymph of fully grown, ligated, and hence starving G. mellonella L. larvae, the label disappeared gradually from glycine and was found in the pool of other free amino acids, both in the haemolymph and in the tissues. The radioactivity of the free amino acid fraction diminished at a rapid rate becoming incorporated into proteins. Ten and 24 hr after injection the proteins contained 24 and 28%, respectively, of the recovered radioactivity. On the other hand, the final products of nitrogen metabolism, i.e. hypoxanthine, xanthine, uric acid and uric acid riboside initially contained only a low % of radioactivity; with time this percentage gradually increased and after 10 hr reached 12% of the total recovered radioactivity, being however always lower than that found in the protein fraction. Radioactive purines and uric acid riboside were found both in the haemolymph and in various tissues; with increasing time after injection they showed a tendency to be transferred towards the excretory organs, where they were removed with the faeces.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of palmitate and stearate by chopped tissue and supernatant preparations

Chopped tissue from developing soybean cotyledons incorporated [1-¹⁴C]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [1-¹⁴C]acetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [1-¹⁴C]acetate or [2-¹⁴C]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [2-¹⁴C]malonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [1-¹⁴C]acetate or [2-¹⁴C]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP's to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.     

Metabolism of [2-14C] acetate to amino acids and proteins in segments of bean seedling roots

Summary ¹⁴C from [2-¹⁴C] acetate was found to be incorporated into soluble and protein amino acids in substantial amounts by bean root apices. The ¹⁴C was spread through a wide range of amino acids in both these fractions. Glutamic acid was found to be heavily labelled with ¹⁴C in both soluble and protein amino acid fractions. The data are discussed in relation to present ideas on transport and utilization of amino acids in root systems.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.