George H. Jones

Phenoxazinone synthase from Streptomyces antibioticus: Purification of the large and small enzyme forms

Abstract Phenoxazinone synthase has been purified approximately 80-fold from 48-h-old cells of Streptomyces antibioticus. The purification procedure involves streptomycin sulfate and ammonium sulfate precipitations, affinity and Bio-Gel chromatography, and glycerol gradient centrifugation. Two forms of the enzyme are purified by these techniques, a large form (L) with a molecular weight of around 900,000 and a small form (S) with an Mr of about 200,000. Electrophoresis of the large and small forms on sodium dodecyl sulfate-polyacrylamide gels reveals the presence of a single polypeptide chain with a molecular weight of around 100,000. Both L and S appear to be distinct molecular forms of the enzyme since rechromatography on Bio-Gel or recentrifugation on glycerol gradients results in retention of the mobility of the form in question. These procedures do not convert L to S or S to L. The purification procedure described has been used for the preparation of phenoxazinone synthase from 12-, 18-, and 48-h cultures of S. antibioticus. These experiments have shown that the relative amounts of L and S which can be isolated depend on the age of the cells used as starting material. As the cultures age, the relative amount of L which is present increases. This result also suggests that L and S are distinct molecular entities. Antibody to a combined L plus S preparation and to L has been raised in rabbits. Anti-L antibody reacts with both L and S and is capable of completely inhibiting phenoxazinone synthase activity in crude extracts of S. antibioticus cells. Thus, the 100,000 Mr subunits of L and S must have some antigenic determinants in common, although they may not be identical in structure. Oxygen consumption during the formation of cinnabarinic acid from 3-hydroxyanthranilic acid was measured with the Clark oxygen electrode. These experiments showed that both L and S catalyze a reaction involving the consumption of 1.5 mol of oxygen per mole of phenoxazinone formed.

Purification of RNA Polymerase from Actinomycin Producing and Nonproducing Cells of Streptomyces antibioticus

DNA-dependent RNA polymerase has been purified approximately 700-fold from 12-h-old cells of Streptomyces antibioticus and 400-fold from 48-h cells. Both enzymes appear nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes possess subunits corresponding to the p, p’, and (Y subunits of Escherichia coli RNA polymerase but no band corresponding to the cr subunit was observed on polyacrylamide gels. Moreover, neither enzyme appears to have (T activity as judged by the rifampicin and heparin challenge assays using T4 DNA as template. In addition to the p, p’, and (Y subunits, electrophoresis of the polymerase from 12-h cells reveals a 45,000 M, protein which is present at a level of 0.40 moYmo1 of p + p’. The polymerases from 12- and 48hS. antibioticus cells differ slightly in their template specificity, with the 48-h polymerase showing a slightly greater preference for calf thymus DNA as compared with several other native DNAs which were tested. Further, the polymerase from 48-h cells was slightly more active with poly (dA-dT) (relative to calf thymus DNA) than was the polymerase from 12-h cells. Neither polymerase was capable of catalyzing actinomycin-resistant transcription.

Effect of marcaine, a myotoxic drug, on macromolecular synthesis in muscle

Abstract The effects of Marcaine (bupivacaine) on RNA and protein synthesis in skeletal muscle have been studied. The drug did not affect RNA synthesis by pieces of rat tibialis anterior at concentrations as high as 0.5% (w/v), nor did it affect cell-free transcription of calf thymus DNA by wheat germ RNA polymerase II. In contrast, Marcaine inhibited protein synthesis by muscle chunks, and also inhibited [ 3 H]leucine incorporation by cell-free components prepared from muscle. Specifically, the drug significantly inhibited aminoacylation of muscle transfer RNA with the amino acids leucine, methionine, lysine and valine (50–90 per cent) at a concentration of 0.5% and also inhibited elongation of polypeptide chains at the same concentration. Marcaine (0.5%) also inhibited aminoacylation of tRNA in cell-free systems derived from rat liver and from murine myeloma RPC-20, but it did not inhibit as strongly as in skeletal muscle. Interestingly, 0.5% Marcaine had no effect on the acylation of tRNA with leucine, methionine, lysine or valine when cell-free components from Escherichia coli were used.

On the efficacy of commonly used ribonuclease inhibitors

Abstract The ability of a number of commonly used inhibitors to inhibit pancreatic ribonuclease has been studied. At ribonuclease concentrations of 10 or 100 g/ml, heparin, polyvinylsulfate and proteinase K, at concentrations reported for their use in the literature, were ineffective in inhibiting RNase digestion of 3 H-uridine labelled RNA from Streptomyces antibioticus . In contrast, macaloid, diethylpyrocarbonate and sodium dodecyl sulfate were all effective inhibitors, with the degree of effectiveness decreasing in the order stated. Further, at inhibitor concentrations which allowed RNase conversion of only 50% of the labelled RNA to acid soluble products, a larger percentage of the acid insoluble digestion products sedimented in the “high molecular weight” range (4–16s) when macaloid was the inhibitor used than when diethylpyrocarbonate was the inhibitor.

Morphological changes accompanying actinomycin production in Streptomyces antibioticus

The fine structure of the actinomycin producing bacterium Streptomyces antibioticus has been studied. The typical appearance of freshly inoculated cells is one of a generally electron dense cytoplasm. As cellular protein synthesis ceases and secondary metabolism (actinomycin biosynthesis) is initiated, a gradual loss of cytoplasmic density occurs, revealing in many cells membrane enclosed vesicles and an extensive cytoplasmic membrane system. Vesicles are seen to be compartmented by cell wall and may be released into the medium by a subsequent breakdown of the cell wall. The data suggest that the appearance of these internal structures is not due to a differentiation of S. antibioticus cells related to the initiation of secondary metabolism, but rather that these vesicles and membrane systems may be normal subcellular structures which become visible only after a loss of cytoplasmic material due to aging of the cells.

Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans

Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

Sensitivity of transcription by purified Streptomyces antibioticus RNA polymerase to actinomycin

Abstract DNA-dependent RNA polymerase has been purified 400-fold from 48 hr old, actinomycin producing cells of Streptomyces antibioticus . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified S. antibioticus polymerase reveals the presence of bands corresponding to the β, β′, and α subunits of Escherichia coli polymerase, but no σ subunit. The purified S. antibioticus polymerase is also associated with a protein component of 145,000 daltons and it is suggested that this protein is produced by limited proteolysis of either the β or β′ subunits. “In vitro” studies have shown that transcription catalyzed by either S. antibioticus or E. coli RNA polymerase is inhibited to about the same extent by actinomycin.

Function of ribosomes and glutamyl-tRNA isoacceptors in protein synthesis in regenerating skeletal muscle

Ribosomes from 8-day-regenerating rat skeletal muscle have been shown to be more active in poly(U)-directed polyphenylalanine synthesis than ribosomes from control muscle. This difference persists after salt washing of the ribosomes and does not appear to be due to the presence of ribonuclease associated with the control ribosome population. Ribosomes from control muscle were also less active than those from regenerates in the nonenzymatic binding of phenylalanyl-tRNA to ribosomes and in the peptidyltransferase reaction. Three glutamyl-tRNA isoacceptors have been isolated from 8-day-regenerating rat skeletal muscle by preparative RPC-5 chromatography of total tRNA charged with [3H]glutamic acid. The two major isoacceptors observed, tRNAgluI and tRNAgluIII, respond to the glutamic acid codons GAG and GAA, respectively. A third, minor glutamyl isoacceptor, tRNAgluII, also responds to the codon GAA. When the three isoacceptors were tested for function in a polysomal cell-free protein synthesizing system, it was found that their relative levels of utilization were essentially identical to their relative abundances. Thus, the tRNA which increases in relative amount after the induction of regeneration, tRNAgluII, is not preferentially utilized for overall muscle protein synthesis.

Time course of changes in protein synthesis in marcaine-induced skeletal muscle regeneration.

The time course of the regeneration of rat skeletal muscle has been examined after injection of the myotoxic drug, Marcaine, to induce regeneration. Muscle wet weight decreases during the initial phase of the regeneration process while the ability of the regenerating muscle to incorporate [35S]methionine into protein, the yield and activity of muscle polysomes and the yield of total and poly(A) + RNA all increase initially. Following the initial changes, these parameters return to near control values by 30 days after Marcaine injection. Theoretical calculations suggest that the changes in polysome yield and activity are sufficient to account for the changes in the ability of muscle fragments to synthesize protein during the regeneration process. The specific activity of total muscle RNA in the wheat germ cell-free system decreases initially during the early stages of the regeneration process. This decrease may reflect the fact that while the yields of both total and poly(A) + RNA increase during the early stages of regeneration, the percentage of the total RNA which is poly(A) + decreases initially.

A protein related to immunoglobulin light chain detected in mouse myeloma cells.

Abstract A protein (denoted L′) which is similar in structure to immunoglobulin light chain has been isolated from the mouse plasma cell tumor, RPC-20. L′ has a molecular weight which is about 6000 daltons less than light chain. The exact nature of the relationship between L′ and light chain has not been established.