Trichloroacetic Acid-insoluble Material Research Articles

Mode of Inhibition of Globin Synthesis by m7G5′;p and m7G5′ppp

The mode of inhibition of rabbit globin synthesis by m7G5'p and m7G5'ppp ("cap analogs") was studied using the rabbit reticulocyte lysate system. The rate of globin synthesis was measured at various concentrations of both f[35S]Met-tRNAf and the cap analogs. The cap analogs were found to inhibit competitively the incorporation of f[35S]Met into hot trichloroacetic acid-insoluble material. Nascent chains prelabelled with f[35S]Met were released at various concentrations of m7G5'ppp. The release of nascent chains was not inhibited by m7G5'p (Suzuki, H. (1976) FEBS Lett. 72, 309) and m7G5'ppp, and it is therefore concluded that the cap analogs inhibit a step of initiation of globin synthesis. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of an 80S/fMet-tRNAf was inhibited by the cap analogs, but not that of a 40S/fMet-tRNAf complex. These data suggest that a factor which is required for the binding of globin mRNA with 40S/fMet-tRNAf complex forms an inactive complex with the cap analogs, so that the cap analogs inhibit globin synthesis.

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- 14C]leucine or δ-amino[4- 14C] levulinic acid (a precursor of phycocyanobilin). The radiolabeled 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 δ-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 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 δ-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.

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [ methyl- 3H]thymidine or [ 14C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[ N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.

The cytokinin-like action of methyl-2-benzimidazolecarbamate on oat leaves and protoplasts

Chlorophyll breakdown in excised oat leaves is retarded by methyl-2-benzimidazolecarbamate, benzimidazole, and kinetin. Protoplasts derived from treated leaves showed lower nuclease activity, increased leucine incorporation, and decreased uridine and thymidine incorporation into trichloroacetic acid-insoluble materials relative to water-treated controls. Methyl-2-benzimidazolecarbamate is more active than benzimidazole in affecting macromolecular synthesis, and both compounds resemble kinetin in their effects on host-plant physiology. Protoplasts can be useful tools in the analysis of the action of pesticides.

Halomethyl-1,2,3-triazole derivatives: a new type of alkylating agent active in mouse transplantable tumors.

A number of halomethyl-1,2,3-triazole derivatives have been tested for their effect on ICR Swiss female mice bearing the Ehrlich carcinoma ascites (ECA) tumor. Two of these compounds, namely 4-bromomethyl-1-(2,3,4,6-tetra-0-acetyl-β-D-glucopyranosyl)-1,2,3-triazole and 4-iodomethyl-1-(2,3,4,6-tetra-0-acetyl-β-D-glucopyranosyl)-1,2,3-triazole, referred to here as compounds H and K, respectively, promoted a significant increase in the median survival time (145% and 195%) when injected IP for 9 consecutive days at doses of 75 mg/kg/injection and 100 mg/kg/injection, respectively. DNA synthesis, as measured by (methyl-3H)thymidine incorporation into trichloroacetic acid-precipitated material, was always inhibited by all the triazole derivatives to a greater extent than the incorporation of (5,6-3H)uridine, (5-3H)proline, and (6-3H)-glucose. Moreover, inhibition of DNA synthesis was complete and irreversible following exposure to selected triazole derivatives. Compounds H and K inhibited the incorporation of di(3H)methyl sulfate by ECA cells. In addition, compound K promoted the release of radioactivity associated with trichloroacetic acid-insoluble material coming from (8-3H)guanosine-prelabeled cells. This release of radioactivity did not occur when cells were prelabeled with (methyl-3H)thymidine. It is concluded from these results that these triazole derivatives act as alkylating agents.

Biosynthesis ofSaccharomyces cerevisiae glycoproteins: Nature of some participating glycolipids

A particulate membrane preparation from Saccharomyces cerevisiae catalyzed the incorporation of mannose from GDP-mannose into lipids that were extractable in chloroform-methanol. One lipid has been previously characterized as dolichyl phosphomannose. Another one was purified by chromatography on silicic acid, DEAE-cellulose and Sephadex LH-20 was found to be alkali unstable. The lipid moiety was shown to be dolichol and the glycosydic part contained mannose, glucose and glucosamine. Radioactive mannose was also incorporated at a slower rate into more polar compounds. They were soluble in chloroform-methanol-water and were seen to liberate neutral oligosaccharides after alkaline hydrolysis. Radioactive mannose was also incorporated into substances which behave chemically as glycoproteins since they were insoluble in organic solvents, water and trichloroactic acid. Pronase treatment of the trichloroacetic acid-insoluble material released water-soluble oligosaccharides. When the particulate preparation which had been extracted with chloroform-methanol at-20 C, was incubated with GDP-(U-14C)mannose, radioactivity was incorporated into glycolipids that were soluble in chloroform-methanol-water and into glycoproteins. This result suggests that at least part of the mannose was transferred to endogenous acceptors independent of dolichyl phosphomannose.

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [(14)C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that (14)C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [(14)C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [(14)C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.

Oxidative Metabolism of the Human Eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane-active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3-6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Oxidative metabolism of the human eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3–6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Maintenance of neurons isolated in bulk from rat brain: incorporation of radiolabeled subtrates

Neurons isolated in bulk from 10- to 15-day-old rat brain can be maintained for 18–24 h. Phase microscopy shows that the cells remain morphologically intact during this time. As a criterion of viability, the incorporation of radiolabeled amino acids and uridine into trichloroacetic acid-insoluble material was selected. The cells are capable of the incorporation both after isolation and after maintenance. The uptake is constant with time, is proportional to substrate concentration, and is inhibited by puromycin and cycloheximide, but not by chloramphenicol. Thus the cells remain viable during this maintenance period. This system may provide a model for the study of the synthesis of specific neuronal components.

Collagen and Non-Collagen Protein Synthesis in the Lungs of Rats Exposed to a Trypsin Aerosol

In rat lungs, 24 h after a 10 min inhalation of a nebulized 1% (w/v) trypsin solution, there was a 25% increase in lung weight. The incorporation of 3H-tryptophane and 2,3-[3H]-proline into trichloroacetic acid insoluble material was decreased although there was no alteration in prolyl hydroxylase activity. Although hydroxyproline formation was decreased, this decrease was probably due to the general decrease in protein synthesis. Ninety-six hours after inhalation of the trypsin solution there was an increase in non-collagen protein biosynthesis. Proline incorporation and hydroxyproline formation were both increased more than the tryptophane incorporation increase at this same time point. These increases were accompanied by an increase in prolyl hydroxylase activity. These experiments indicate that major changes in protein biosynthesis occur in lung tissues after inhalation of proteolytic enzymes and demonstrate the temporal biochemical changes which occur in lung injury.

Incorporation of l-azetidine-2-carboxylic acid into hemoglobin S in sickle erythrocytes in vitro

l-Azetidine-2-carboxylic acid, the naturally occurring lower homologue of l-proline, is incorporated into hemoglobin S (sickle hemoglobin) in vitro. Sickle erythrocytes from patients with sickle cell anemia incubated with l-[ 3H]azetidine-2-carboxylate synthesized radiolabeled hemoglobin which when isolated from cell lysates, co-chromatographed with hemoglobin S on DEAE-cellulose columns. The α β ratio of azetidine carboxylate incorporation into the globin chains of sickle hemoglobin was 0.94, which is consistent with the presence of four proline residues in each polypeptide chain. Incorporation of azetidine carboxylate into hot trichloroacetic acid-insoluble material in sickle erythrocytes indicated that the homologue was present in the polypeptide backbone of the globin chains of sickle hemoglobin. Amino acid analysis of the hot trichloroacetic acid-insoluble material from sickle erythrocytes which had been incubated with radiolabeled azetidine carboxylate indicated that 75% of the radioactivity could be accounted for as intact homologue while 20% of the radioactivity co-chromatographed with alanine. These results suggest that azetidine carboxylate is incorporated unaltered into hemoglobin S in addition to being metabolized to alanine in sickle erythrocytes prior to incorporation into protein. The kinetics of thermal precipitation of hemoglobin S (oxygen ligand) into which radioactive azetidine carboxylate or radioactive proline had been incorporated in vitro is identical. This observation, together with the behavior of hemoglobin S and the globin chains from hemoglobin S containing azetidine carboxylate during ion-exchange chromatography, indicates that homologue replacement of prolyl residues does not significantly alter the overall charge or stability of the hemoglobin S tetramer. Azetidine carboxylate did not inhibit uptake of radiolabeled proline by sickle erythrocytes suggesting that the homologue does not adversely affect amino acid transport in these cells.

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.

Interferon activity produced by translation of human interferon messenger RNA in cell-free ribosomal systems and in Xenopus oöcytes.

Translation of messenger RNA isolated from poly(rI)-poly(rC)-induced human fibroblasts in cell-free ribosomal systems and in Xenopus oocytes resulted in the production of biologically active proteins that had the properties of human fibroblast interferon. The translation in the oocytes was much more efficient, giving approximately 500 times higher titers of interferon activity than the cell-free systems. A control messenger RNA isolated from noninduced human fibroblasts, did not code for interferon synthesis in these systems. Both messenger RNA preparations stimulated [3H]amino-acid incorporation into trichloroacetic acid-insoluble material. The radioactive products and their immunoprecipitates were electrophoresed on polyacrylamide gels under denaturing conditions. The products resulting from the translation of the control (uninduced) messenger RNA in oocytes contained a major protein of approximately 45,000 molecular weight. The messenger RNA isolated from poly(rI)-poly(rC)-induced cells stimulated the synthesis of an additional 25,000 molecular weight protein that electrophoresed in the same position as human fibroblast interferon. These results suggest that human fibroblast interferon was synthesized by the translation of its messenger RNA in Xenopus oocytes and in cell-free ribosomal systems.

Cytoplasmic synthesis of soluble and mitochondrial malate dehydrogenase isozymes in maize

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

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant. The incorporation of L-[U- 14C]proline and L-[ 3H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.

Effect of phenylalanine and its metabolites on the metabolism of leucocytes and lymphocytes.

1. The pathogenesis of the mental retardation in phenylketonuria remains obscure. Leucocytes have proved of value in the study of other inborn errors of metabolism. The lymphocyte is a suitable model cell for the study of mammalian metabolism, because of its ability to divide in vitro in response to various stimuli. 2. We have examined the effects of phenylalanine, phenylpyruvate, phenyl-lactate and phenylacetate on the human leucocyte and the resting and phytohaemagglutinin-stimulated rabbit lymphocyte. 3. Phenylpyruvate and phenyl-lactate reduced acetate incorporation into leucocyte lipid by 38% and 48% respectively. Only phenyl-lactate reduced acetate incorporation into the resting and stimulated lymphocyte, by 20% and 34% respectively. 4. Glucose incorporation into leucocyte lipid was unaffected by phenylalanine, phenylpyruvate and phenyl-lactate. Only phenyl-lactate inhibited (46%) the production of CO2 from glucose. 5. Phenylalanine and leucine incorporation into trichloroacetic acid-insoluble material of resting and stimulated lymphocytes was inhibited by phenyl-lactate (10-42%), phenylpyruvate (27-57%) and phenylacetate (19-39%). 6. Uridine incorporation into resting and stimulated cells was inhibited by phenyl-lactate (22-26%), phenylpyruvate (42-52%) and phenylacetate (20%). 7. Thymidine incorporation into resting lymphocytes was reduced by phenyl-lactate, phenylpyruvate, phenylacetate and phenylalanine by 12-26%. Incorporation into the stimulated cell was inhibited by phenylpyruvate and phenyl-lactate (90%) and phenylacetate (66%). 8. Phenylalanine inhibited lymphocyte pyruvate kinase and phenylpyruvate inhibited citrate synthetase. 9. These results are compared with published data relating to experimental hyperphenylalaninaemia and the effects of these metabolites on nervous tissue in vitro.

Drug-induced suppression of phospholipid synthesis in hela cells by inhibition of choline uptake

2-(4-Anisidino)-4,6-bis(2-(diethylmethylammonium)ethylamino)-1,3,5-triazine diiodide was found to inhibit specifically the incorporation of [ 14C]-choline into cold 5% trichloroacetic acid-insoluble materials in HeLa cells without affecting other vital areas of cellular metabolism. Further studies indicated that this drug did not affect any of the intracellular reactions leading to phosphatidylcholine formation; instead, the inhibition of lecithin synthesis was due primarily to the suppression of choline transport through the cytoplasmic membrane. The level of inhibition of the latter process was comparable to that observed for the inhibition of choline incorporation into cold 5% trichloroacetic acid-insoluble precipitates in whole cells.

Chloramphenicol resistant mitochondrial protein synthesis in Crithidia fasciculata

Growth of Crithidia fasciculata, a heme-requiring flagellated protozoan, was not affected by the presence of chloramphenicol in concentrations as high as 4 mg/ml. Amino acid incorporation by a mitochondrial fraction, containing both mitochondria and kinetoplasts, was resistant to chloramphenicol and cycloheximide, but sensitive to puromycin. The postmitochondrial supernatant (27,000 g) also incorporated [ 14C]leucine into trichloroacetic acid insoluble material, but this incorporation was inhibited by cycloheximide. Chloramphenicol did not affect this system. Treatment of the mitochondrial fraction with digitonin enhanced the rate of [ 14C]leucine incorporation, but did not render the preparation sensitive to chloramphenicol. Electron microscopic examination of the digitonin treated mitochondrial fraction revealed the presence of ghosts. Separation of kinetoplasts from mitochondrial vesicles was achieved by discontinuous sucrose gradient centrifugation. Both the kinetoplast and mitochondrial fractions incorporated [ 14C]leucine at about the same rate. No inhibition of incorporation by chloramphenicol was observed with either fraction.

The involvement of extracellular enzymes in the metabolism of Bdellovibrio.

1. Three host-independent (H-I)Bdellovibrio species are stimulated in growth by addition to the medium of cell-free host extracts. A mixture of fifteen labeled amino acids was used to follow incorporation of counts into trichloroacetic acid (TCA) insoluble material. No label was recovered in the nucleic acids. 2. LabeledEscherichia coli RNA, protein, andAeromonas DNA preparations were hydrolyzed byBdellovibrio extracellular enzymes. The hydrolyzed protein or nucleic acids were added to a host free medium and inoculated withBdellovibrio. The amount of uptake and TCA precipitable counts in stationary phase cells was then determined. From these studies it has been demonstrated thatBdellovibrio extracellular enzymes hydrolyze host macromolecules to products which are subsequently used in the biosynthetic pathways ofBdellovibrio. 3. It is also suggested thatBdellovibrio cannot synthesize their own nucleotide bases, since neither14C-glycine nor3H-aspartic acid was incorporated into hot TCA soluble material.