Origin of the sarcosine molecules of actinomycins

1. Streptomyces V–187 produces on minimal medium a mixture composed mainly of actinomycin C₁ (actinomycin D) and actinomycin A₁ (actinomycin I). If sarcosine is added to the medium, the micro-organism produces, in addition to actinomycins C₁ and A₁, actinomycin F₈ (actinomycin II) and actinomycin F₉ (actinomycin (III), characterized by the substitution by sarcosine of one or both the proline molecules present in actinomycin C₁. 2. Exogenous sarcosine seems to be incorporated as such by Streptomyces V–187 only in the sarcosine molecule(s) that replace proline in the actinomycins of the F group, whereas, for the synthesis of the other sarcosine molecules, the amino acid is first demethylated to glycine. 3. The incorporation of sarcosine and glycine into actinomycin by Streptomyces antibioticus appears to follow a similar pattern, except that a portion of the methyl group produced in the degradation of sarcosine is utilized as a source of the methyl groups of the antibiotic. This explains the previously reported lack of cross-dilution between glycine and sarcosine observed in the incorporation of these amino acids into actinomycin.     

3-hydroxy-5-methylproline, a new amino acid identified as a component of actinomycin Z1.

3-Hydroxy-5-methylproline has been identified in hydrolysates of actinomycin Z₁ by ion-exchange and paper chromatography, paper electrophoresis, gas chromatography and mass spectrometry in comparison with the synthetic compound. The stereochemistry of this amino acid is under investigation. The amino acid composition of actinomycin Z₁ thus consists of threonine, hydroxythreonine, D-valine(2), 4-oxo-5-methylproline, 3-hydroxy-5-methylproline, sarcosine(2), N-methylalanine and N-methylvaline.     

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.     

Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.     

Variation in amino acid composition of cytochrome c of species in the fungal genusUstilago

Cytochrome c was extracted and purified from nine species of the genusUstilago, representing five pathogen for monocotyledonous and four for dicotyledonous host species. The amino acid compositions of acid hydrolysates of cytochrome c from these species were compared and divergence values were calculated for all pairs of species. Aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, leucine, phenylalanine, lysine, and arginine were the most variable amino acids for both dicot and monocot pathogens. Differences in lysine, glutamic acid, alanine, and histidine distinguished most monocot and dicot pathogens. The dicotyledonous pathogens had fewer glutamic acid and additional alanine and histidine residues. Divergence values for cytochrome c were generally highest between species of dicotyledonous and monocotyledonous hosts and lowest for species within the monocotyledonous group. Pairs of species in the dicotyledonous group gave values only slightly lower than those for pairs with species from different groups.     

A new method for the preparation of delta 1-pyrroline 5-carboxylic acid and proline.

Hydroxylysine was oxidized with sodium metaperiodate and the major product characterized as Δ¹-pyrroline 5-carboxylic acid by the visible spectrum of its o-aminobenzaldehyde complex and by its reduction to proline by subsequent treatment with sodium borohydride. The reduction product was positively identified as proline by proton nuclear magnetic resonance spectroscopy and mass spectral analysis. The pH dependence for the preparation of Δ¹-pyrroline 5-carboxylic acid was determined by a study of the yields of proline obtained by variation of the pH values of the oxidative step. These observations support the hypothesis of intramolecular cyclization of α-aminoglutaric γ-semialdehyde as the second step in the reaction mechanism.     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

Amino acid metabolism during flight in tsetse flies.

Experiments carried out using teneral Glossina pallidipes indicate that flight can continue for at least 4 to 7 min after the thoracic proline reserves have fallen to low levels, suggesting that some other energy source is available. Earlier work suggests that alanine formed during flight is transported from the thorax to the abdomen where proline is resynthesized. Injection experiments using ¹⁴C alanine confirm that the transport mechanism does occur, that it is enhanced by flight, and that alanine is more rapidly incorporated into glutamate and proline in the abdomen than in the thorax. An analysis of published work shows that there is evidence for the involvement of residual blood meal amino acids even in the early stages of flight and supports the suggestion that they are of importance in prolonging flight. A decline in amino nitrogen during the early stages of flight is consistent with the action of glutamate dehydrogenase at this time. The poor flight durations in teneral flies may be due both to the low proline levels and to the absence of the residual blood meal. Very high energy consumptions are noted and appear to be related to the abnormally large musculature necessary for the fly's haematophagous and viviparous habits.     

New internal standards for basic amino acid analyses

Eight derivatives of cysteine and penicillamine with 2- and 4-vinyl-pyridine, p-nitrobenzyl bromide, and p-nitrostyrene were evaluated as potential internal standards for the short and long (physiological) basic columns in amino acid analysis by ion-exchange chromatography. S-β-(4-pyridylethyl)-dl-penicillamine (4-PEP) was found to have an advantage over the previously proposed S-β-(4-pyridylethyl)-l-cysteine (4-PEC) since the elution position of 4-PEP on the short basic column is insensitive to minor changes in pH of the eluting citrate buffer. 4-PEP was found to be stable to acid hydrolysis as used for proteins and its recovery from protein hydrolysates was unaffected by the presence of starch during hydrolysis. However, an extra 14 min is required to elute 4-PEP on the short column.Of the eight compounds studied, six appcar suitable as internal standards on the physiological (long) column. These elute in widely differing positions between histidine and arginine, thus offering a choice of internal standards for special analysis on the basic long column.     

New actinomycin from Actinomyces sp. No. 2

A new actinomycin was isolated from a mixture of actinomycins formed by Actinomyces sp. No. 2, an organism producing auranthin, an actinomycetous antibiotic. The peptide chains of the new actinomycin contain such amino acids as threonine, valine, proline and sarcosine in a ratio of 2 : 4 : 2 : 2. N-Methyl-valine characteristic of all actinomycins is replaced in position 5 of both pentapeptide chains of the new actinomycin by valine. The new actinomycin is actinomycin D undermethylated in position 5 by valine. When the growing culture of Actinomyces olivobrunneus producing actinomycin D was exposed to sulfadimesine, an inhibitor of biological methylation, production of actinomycin D0 (sarcosine replaced by glycine in one of the pentapeptide chains) markedly increased, which indicated impairment of the glycine residue methylation. Still, no impairment of the valine residue methylation in position 5 of the pentapeptide chains was observed an no actinomycin with N-methyl-valine replaced by valine was formed.     

Developmental control of 2-aminoisobutyric acid transport by 7-and 14-day chick heart cell aggregates. Roles of insulin and amino acids.

Cell aggregates cultured from 7-day embryonic avian heart showed a spontaneous increase in A-system 2-aminoisobutyric acid transport when placed in protein-free and amino acid-free buffer for 3 hr. The apparent V_max increased from 4.0 to 9.9 nmoles/μl of intracellular fluid volume/10 min in 3 hr. l-Proline (5 mM), an amino acid transported primarily by the A system, prevented this rise, but l-phenylalanine, primarily an L-system substrate, had no effect. Actinomycin, puromycin, and cycloheximide (55 μM) also prevented the time-dependent increase in transport. In contrast, cell aggregates cultured from 14-day embryonic heart exhibited a decrease in apparent V_max during the 3-hr incubation, from 8.3 to 3.3 nmoles/μl of intracellular volume/10 min. l-Proline, but not l-phenylalanine, enhanced this decrease in A-system transport. The percentage proline inhibition of transport was reduced by actinomycin or cycloheximide (55 μM) at both ages. Insulin stimulated A-system transport at identical half-maximal concentrations of 18 nM at 7 and 14 days of embryonic development. In the presence of cycloheximide at 7 days of age, insulin prolonged the half-life of transport activity twofold. However, at 14 days, cycloheximide reduced the insulin response by 88% [Elsas, L. J., Wheeler, F. B., Danner, D. J., and DeHaan, R. L. (1975). J. Biol. Chem.250, 9381–9390]. l-Proline or actinomycin reduced both basal and insulin-stimulated transport by 7-day cell aggregates, but neither reduced the percentage insulin stimulation. We conclude that inherent developmental control(s), A-system amino acids, and insulin regulated the maximal velocity of A-system transport by controlling the biological turnover of transport protein(s). l-Proline decreased the existing synthesis of transport protein(s) at both ages. The predominant effect of insulin shifted from a posttranslational level at 7 days to a synthetic level by 14 days of embryonic development. Seven-day cell aggregates spontaneously increased synthesis in the absence of A-system amino acids, but 14-day cell aggregates required hormonal stimulation to shift the balance from degradation to synthesis of transport protein(s).     

Inhibition by glucose of the synthesis of proline from glutamic acid

While in the absence of glucose, proline is not a required amino acid, in the presence of glucose the growth of Micrococcus pyogenes var. aureus in amino acid medium is proportional to the concentration of proline when all other amino acids and growth factors are present in amounts adequate for optimal growth. The data presented here and the ideas prevailing in the literature indicate that glutamic acid is a precursor of proline. Glucose inhibits the conversion of glutamic acid into proline, which in turn causes failure of growth. Thus, 1 μg. and 10 μg. glucose/ ml. cause 50% and 100% inhibition, respectively, of the growth dependent on the synthesis of proline. One μg. proline antagonizes completely the inhibition in the presence of 5,000 μg. glucose/ml.One μg. glycerol, 100 μg. pyruvate, 250 μg. lactate, or 100 μg. α-glycerophosphate/ml., individually, cause from 25 to 50% inhibition of the growth dependent on the synthesis of proline from glutamic acid. Five thousand μg./ml. either of malic, succinic, fumaric, α-keto-glutaric, cis-aconitic acid, or dihydroxyacetone, or 500 μg. citric acid/ml. fails to cause inhibition.Pyrrolidone carboxylic acid was found to substitute for glutamic acid but not for proline in tests with M. pyogenes var. aureus. Also, seven proline-less mutant strains of Escherichia coli were unable to utilize pyrrolidone carboxylic acid in place of proline. No evidence was obtained to indicate that pyrrolidone carboxylic acid could serve as a direct precursor of proline.     

Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication.

The number of proline residues in a protein should have very marked consequences for the rates of protein unfolding and refolding according to the model proposed by Brandts et al. (1975). Kinetic simulations of this model indicate that the half-time for refolding of a polypeptide chain with 20 proline residues should be greater than 10 minutes and should increase by about an order of magnitude for each additional 10 proline residues. Various means are considered by which the rate of protein folding in vivo and in vitro might be increased.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Antiviral Effect of Diammonium Glycyrrhizinate on Cell Infection by Porcine Parvovirus

The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: β-naphthyl amide derivatives of l-anomers of leucine, lysine, proline, glycine–proline, and phenilalanine–proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile.     

Purification and properties of proline reductase from Clostridium sticklandii.

Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.     

Involvement of Proline Oxidase (PutA) in Programmed Cell Death of Xanthomonas

Xanthomonas campestris strains have been reported to undergo programmed cell death (PCD) in a protein rich medium. Protein hydrolysates used in media such as nutrient broth comprise of casein digest with abundance of proline and glutamate. In the current study, X. campestris pv. campestris (Xcc) cells displayed PCD when grown in PCD inducing medium (PIM) containing casein tryptic digest. This PCD was also observed in PCD non-inducing carbohydrate rich medium (PNIM) fortified with either proline or proline along with glutamate. Surprisingly, no PCD was noticed in PNIM fortified with glutamate alone. Differential role of proline or glutamate in inducing PCD in Xcc cells growing in PNIM was studied. It was found that an intermediate product of this oxidation was involved in initiation of PCD. Proline oxidase also called as proline utilization A (PutA), catalyzes the two step oxidation of proline to glutamate. Interestingly, higher PutA activity was noticed in cells growing in PIM, and PCD was found to be inhibited by tetrahydro-2-furoic acid, a competitive inhibitor of this enzyme. Further, PCD was abolished in Xcc ΔputA strain generated using a pKNOCK suicide plasmid, and restored in Xcc ΔputA strain carrying functional PutA in a plasmid vector. Xanthomonas cells growing in PIM also displayed increased generation of ROS, as well as cell filamentation (a probable indication of SOS response). These filamented cells also displayed enhanced caspase-3-like activity during in situ labeling using a fluorescent tagged caspase-3 inhibitor (FITC-DEVD-FMK). The extent of PCD associated markers such as DNA damage, phosphatidylserine externalization and membrane depolarization were found to be significantly enhanced in wild type cells, but drastically reduced in Xcc ΔputA cells. These findings thus establish the role of PutA mediated proline oxidation in regulating death in stressed Xanthomonas 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.