Hot Trichloroacetic Acid-insoluble Material Research Articles

Effect of Norspermidine and Its Related Triamines on the Cell‐Free Polyphenylalanine Synthesizing System from Vibrio parahaemolyticus

The effect of norspermidine and its structurally related triamines on the cell-free polyphenylalanine synthesizing system from Vibrio parahaemolyticus was examined in connection with the requirement of the system for monovalent cation. In the absence of norspermidine, the maximal incorporation of [14C]phenylalanine into hot trichloroacetic acid insoluble material was observed under ionic conditions of 12 mM Mg2+ and 50 mM NH4+. K+ could partially substitute for NH4+, but Na+ could not. The addition of norspermidine to the polyphenylalanine synthetic reaction mixture not only lowered the optimal Mg2+ concentration, but it also stimulated the polyphenylalanine synthesis up to 2-fold with no significant increase in misincorporation of [14C]leucine. Other triamines having one or two methylene chains more than norspermidine were also effective in eliciting these effects. Furthermore, Na+ could not support the polyphenylalanine synthesis even in the presence of norspermidine and, on the contrary, inhibited the polyphenylalanine synthesis induced by NH4+ regardless of whether norspermidine was present or not. These findings are discussed in comparison with the properties of other bacterial cell-free systems.

Glucosylation of glycoproteins by mammalian, plant, fungal, and trypanosomatid protozoa microsomal membranes

An assay for UDP-Glc:glycoprotein glucosyltransferase was developed. Incubation of rat liver microsomes with UDP-[14C]Glc led to the formation of hot trichloroacetic acid insoluble material identified as protein-linked Glc1Man7-9GlcNAc2. Addition of 8 M urea-denatured thyroglobulin to the incubation mixtures stimulated up to 10-12-fold the formation of the same compounds but only in the presence of detergents. Native thyroglobulin was ineffective. Several experiments indicated that the stimulation was due to the transfer of glucose residues from UDP-Glc to high-mannose oligosaccharides in urea-denatured thyroglobulin and that this transfer reaction did not involve dolichol mono- or diphosphate derivatives as intermediates. The glycoprotein glucosylating activity was mainly located in the endoplasmic reticulum and could glucosylate glycopeptides derived from the digestion of thyroglobulin with an unspecific protease. Glucosylation of oligosaccharides in those glycopeptides occurred, however, at a rate at least 2 orders of magnitude slower than that of the same compounds in urea-denatured thyroglobulin. Tryptic digestion of urea-denatured thyroglobulin did not affect its glucosylation rate. The structure of Glc1Man9GlcNAc2 linked to urea-denatured thyroglobulin was identical with that of Glc1Man9GlcNAc2-P-P-dolichol. The assay of UDP-Glc:glycoprotein glucosyltransferase allowed detection of the activity in microsomal membranes in which endogenous acceptors appeared to be absent or almost absent, such as those derived from mung bean, Mucor rouxii, Crithidia fasciculata, and Trypanosoma cruzi cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores.

Incubation of purified vaccinia virus with gamma-(32)P-adenosine triphosphate resulted in the incorporation of (32)P into hot trichloroacetic acid-insoluble material. Enzymatic activity was completely dependent on the addition of divalent cations and was stimulated by nonionic detergents and dithiothreitol. Chemical studies demonstrated that serine and threonine residues of 15,000 molecular weight viral polypeptides were phosphorylated. In contrast, the major structural proteins were not phosphorylated or were phosphorylated to a much lesser extent. Added histones and protamine, but not serum albumin, casein, or phosvitin were phosphorylated by the partially disrupted vaccinia virus preparations. The protein kinase was tightly associated with vaccinia virus particles since the specific enzymatic activity remained constant during the final steps of virus purification, the specific activities of many different preparations of virus were similar, and the enzymatic activity cosedimented with vaccinia virus during rate zonal sucrose gradient and potassium tartrate gradient equilibrium centrifugations. Controlled degradation of vaccinia virus, with nonionic detergents and dithiothreitol, indicated that both the protein kinase and the specific phosphate acceptor proteins were located in the virus core.

Anti-microbial drugs and RNA

1. 1. The behaviour of the pulse-labelled RNA and of protein synthesis in a thermophilic Bacillus in the presence of actinomycin D, proflavine or ethidium has been examined. 2. 2. All concentrations of actinomycin above the minimum which prevented RNA synthesis gave breakdown of pulse-labelled RNA. The rate of breakdown and the fraction of the labelled RNA which did not breakdown were independent of the concentration of actinomycin. 3. 3. Breakdown of pulse-labelled RNA could also be demonstrated using proflavine or ethidium. The half-life of the breakdown with these two drugs varied linearly with drug concentration. High concentrations of either drug protected the pulse-labelled RNA from breakdown. 4. 4. The rate of loss of the capacity to incorporate radioactive leucine into hot trichloroacetic acid-insoluble material in the presence of actinomycin was independent of the concentration of actinomycin. With proflavine or ethidium this rate of loss increased with increasing drug concentration. 5. 5. It was concluded that proflavine and ethidium inhibit both the breakdown of pulse-labelled RNA and the functioning of the protein-synthesising complex, whereas actinomycin D does not. This difference is probably due to the ability of the former two drugs to complex with RNA.

Protein synthesis in Artemia salina embryos : I. Studies on polyribosomes

A study of polyribosomes from encysted embryos of the crustacean Artemia salina has been carried out by sucrose density gradient centrifugation. Although no discrete polyribosome peaks were detected in extracts from hydrated-dormant gastrulae, a small amount of ribonuclease-sensitive material sedimenting below the ribosomes was present. Formation of small polyribosomes has been detected within 5 minutes after the dormant gastrulae were permitted to resume metabolism and development by incubation at 30°C. The increase in polyribosome content which occurred with further incubation appeared to result chiefly from the formation of heavier polyribosomes. Using NaH 14CO 3 as a precursor, protein synthesis has been detected in intact gastrulae within the first 15 minutes after the end of dormancy. Polyribosomes were shown to be functional by their in vitro incorporation of leucine- 14C into hot trichloroacetic acid-insoluble material. An evaluation has been made of the method used for the study of polyribosomes in Artemia.

The arginine transfer reaction

The incorporation of arginine into hot trichloroacetic acid-insoluble material by a 150 000 · g supernatant fraction from sheep thyroid cytoplasm has been studied. 1. 1. Incorporated arginine contains a free α-amino group as judged by the fluorodinitrobenzene reaction. It can be recovered as free arginine after acid hydrolysis or digestion by pronase. 2. 2. Arginyl-tRNA is an intermediate and an enzyme has been partially purified which transfers arginine from tRNA to protein. This enzymatic transfer displays a partial requirement for a non-enzymatic protein. Boiled supernatant fraction, albumin and thyroglobulin fulfill this function, whereas hemoglobin, myoglobin, cytochrome c and polyaspartic acid do not. 3. 3. The transfer has a pH optimum between 9.0 and 9.8 and is highly dependent upon the presence of mercaptoethanol and upon the ionic strength of the reaction mixture. It does not require ribosomes, Mg 2+ nor GTP and it is neither inhibited by puromycin nor stimulated by poly(A, G). 4. 4. A similar system for the incorporation of arginine exists in the soluble fraction of rabbit liver cytoplasm. The data are most consistent with an addition reaction in which arginine is transferred from tRNA onto certain preexisting proteins.

Protein synthesis by extracts from subcellular particles of sheep thyroid

Protein synthesis has been studied in extracts obtained by disruption of particles sedimenting between 700 and 10,000 g. (a) Aminoacyl sRNA synthetase activities can be demonstrated in such extracts. At limiting concentrations of sRNA they attach about twice as much l-phenylalanine and l-leucine but somewhat less valine per unit sRNA than does a corresponding supernatant fraction from the cytoplasm. Mixing experiments indicate that, under these conditions, the cytoplasm contains a heat-stable inhibitor which reduces the amount of l-leucine attached to sRNA by extracts from the particles. (b) Extracts from the particles also contain the enzymes required to transfer phenylalanine from sRNA into protein. This reaction is partially GTP-dependent and is stimulated by polyuridylic acid. It displays an absolute requirement for ribosomes as does incorporation into protein starting with free phenylalanine. Ribosomes isolated from the particles by treatment with deoxycholate are active. (c) Extracts from the particles can incorporate l-glutamic acid into hot trichloroacetic acid-insoluble material in the absence of ribosomes. The product is rendered acid-soluble by treatment with trypsin. This reaction does not require magnesium ions and is not inhibited by puromycin. It is also not inhibited by RNAase, indicating that glutamyl sRNA is probably not an intermediate.

Genetic code and streptomycin: Their relation to ribosomal subunits

The following combinations of synthetic polynucleotide and aminoacyl-sRNA caused binding of the aminoacyl-sRNA to the 30-S ribosomal subunits in the absence of 50-S ribosomal subunits: leucyl-sRNA (poly (U 8, A, C), poly (U 8, G, C), poly (U 4, G), poly (U 4, A), poly (U)), seryl-sRNA (poly (U 8, A, C), poly (U 8, G, C)), isoleucyl-sRNA (poly (U 4, A), poly (U 8, A, C), valyl-sRNA (poly (U 4, G), poly (U 8, G, C)), tyrosyl-sRNA (poly (U 8, A, C)), lysyl-sRNA (poly (A 3, G)). Streptomycin does not stimulate the binding of isoleucyl-sRNA to 30-S subunit-template complex, while it stimulates the binding of isoleucyl-sRNA to ribosome-template complex. When ribosomes were dissociated into their subunits and the subunits were reassociated by raising the concentration of Mg 2+, streptomycin did not stimulate the binding of isoleucyl-sRNA to these reassociated ribosomes in the presence of poly (U), poly (U 4, A), or poly (U 8, A, C). The incorporation of [ 14C]isoleucine into hot trichloroacetic acid-insoluble material in the presence of these synthetic polynucleotides and 2·10 −2 M Mg 2+ by the reassociated ribosomes was greater than that by the non-treated ribosomes. Streptomycin did not influence the incorporation of isoleucine into polypeptide by reassociated ribosomes under the direction of poly (U 4, A), while it stimulated the incorporation by non-treated ribosomes under the same conditions.

Membrane-associated protein synthesis in Streptococcus faecalis

1. Cell-free extracts of Streptococcus faecalis were separated centrifugally into a ribosomal, a washed membrane, and several intermediate fractions. All fractions incorporate labeled amino acids into hot trichloroacetic acid-insoluble material. The requirements for amino acid incorporation are similar for all fractions, and electro-phoresis of the reaction products gives identical polypeptide peaks. 2. All particulate fractions contain phospholipid, and the level of amino acid incorporation is proportional to phospholipid content. No qualitative differences are found in the phospholipid composition of the various fractions. 3. Incorporation by cell fractions is also proportional to RNA content, except for the ribosomal fraction and a first membrane washing, where “excess” RNA appears to be present. On sucrose density gradients of ribosomal fractions the bulk of the “excess” RNA is isolated as a broad peak with a sedimentation coefficient of 80–150 S. On mild ribonuclease (EC 2.7.7.16) treatment this peak dissociates into 30-20 S ribosomal subunits. 4. In addition to large 30-20 S ribosomal aggregates, crude extracts of S. faecalis contain large polyribosomal aggregates. The polyribosomes, consisting of 30-S and 50-S subunits are released from membranes on exposure to deoxycholate. The ribosomes appear to be bound to membranes through the 50-S subunit. These membrane-bound polyribosomes are ribonuclease-sensitive, but are not degraded on incubation with a soluble fraction of S. faecalis. 5. It is concluded that in S. faecalis the bulk of the ribosomes are in a high state of aggregation. In fractionated extracts, protein synthesis is a function of the concentration of membrane-associated polyribosomes. Possible implications of this finding on mechanisms of protein synthesis are discussed.