Burton M. Pogell

Biosynthesis of puromycin in Streptomyces alboniger: regulation and properties of O-demethylpuromycin O-methyltransferase.

Mechanisms for regulation of puromycin biosynthesis in Streptomyces alboniger were studied by measuring the levels of S-adenosyl-l-methionine:O-demethylpuromycin O-methyltransferase. The enzyme was released in soluble form from mycelia by 3 to 5 min of sonication at 4 C. Maximal specific activities of 0.7 and 0.1 nmol/min per mg of protein were found in cells grown in corn steep liquor-corn starch and Hickey-Tresner media, respectively. In both media, the O-methyltransferase activity rose from low levels to a maximum during midlogarithmic growth and then declined or disappeared completely (in Hickey-Tresner medium) during stationary phase. Either glucose (1%) or ethidium bromide (5 μM) reduced O-methyltransferase formation to very low levels with no effect on overall growth. Complete glucose repression of antibiotic formation occurred on agar. Cells grown in the presence of ethidium bromide continued to produce low enzyme levels after regrowth in the absence of dye, but formed normal amounts of puromycin on Hickey-Tresner agar. The O-methyltransferase, either crude or purified, was rapidly inactivated at 37 C. Each substrate alone, or both together at lower concentrations, protected against this loss of activity. Puromycin inhibited the transferase. Regulation of O-methyltransferase synthesis in S. alboniger includes (i) induction early in growth that is susceptible to catabolite repression and differential inhibition by ethidium bromide, and (ii) protection of the enzyme from inactivation by increased intracellular levels of its substrates. The O-methyltransferase was purified 30- to 40-fold by a combination of protamine sulfate precipitation, ammonium sulfate fractionation, adsorption and gradient salt elution from diethylaminoethyl-cellulose and Sephadex G-200 gel filtration. The enzyme was very unstable, even at low temperatures, upon purification beyond the salt fractionation step, but was stabilized by the addition of S-adenosyl-l-methionine during later stages of purification.

Variability in the catalytic and allosteric properties of rabbit liver fructose 1,6-diphosphatase: The presence of tryptophan in homogeneous enzyme*

Summary Rabbit liver fructose 1,6-diphosphatase purified to homogeneity by the procedure of Sarngadharan et al. (1) has been found to contain tryptophan. The basis for variability in tryptophan content and catalytic and regulatory properties of different preparations of the enzyme is discussed. Variable proteolysis during isolation is presumed to be the major factor.

A simple, one-step assay for methyltransferase activity: Direct partitioning of radioactive product into scintillation fluid

Abstract A simple, one-step procedure is described for measurement of the enzymatic transfer of radioactive methyl groups from S -adenosyl- l -methionine to O -demethylpuromycin. Reactions are carried out in small, stoppered scintillation vials and terminated by the addition of 0.1 m sodium borate-5 m NaCl (pH 9) buffer and scintillation fluid. The vials are then shaken to extract radioactive pyromycin into the organic phase and directly counted.

Pamamycin inhibits nucleoside and inorganic phosphate transport in staphylococcus aureus

Summary Pamamycin is a stimulator of aerial mycelia formation in Streptomyces alboniger and a new antibiotic active against many microorganisms. Studies in Staphylococcus aureus showed that pamamycin inhibited the uptake of nucleosides and inorganic phosphate, as well as purine and pyrimidine bases. Under the same conditions, other cellular functions including protein and cell wall synthesis, amino acid uptake, glucose utilization and DNA and RNA polymerase activities were not affected. The existence of respiration-dependent transport systems for nucleosides in membrane vesicles of S. aureus also was demonstrated. Pamamycin inhibited membrane transport of all the nucleosides tested. The inhibition of the above membrane-associated functions was probably due to the membrane-binding property of this antibiotic.

Quantitative estimation of newly synthesized radioactive proteins: Use of minicolumns of antibody embedded in agarose gel or immobilized on Sepharose

Abstract Immunochemical methods for the quantitative determination of radioactive proteins synthesized by chick embryo hepatocyte cultures are described. Cell culture medium containing secreted labeled serum proteins was concentrated and applied to agarose gels containing rabbit anti-chicken serum. Nonspecific binding in control gels was reduced to less than 2% of applied counts under conditions where more than 60% of the nondialyzable counts were precipitated in the presence of antibody. Labeled cytosol fructose-1,6-bisphosphatase (Fru-P2ase) from cell sonicates was selectively adsorbed onto columns containing Sepharose-immobilized anti-chicken liver Fru-P2ase. Radioactivity bound on these columns was eluted with 1% sodium dodecyl sulfate for electrophoretic analysis. Addition of dialyzed horse serum was used in both cases to minimize nonspecific binding. These techniques provide simple alternatives to direct immunoprecipitations in solution where nonspecific binding of radioactivity may be unacceptably high.

Occurrence of new metabolites containing N6,N6-dimethyladenine in spores of Streptomyces alboniger☆

Abstract Methods are described for demonstrating the presence of metabolites containing N6,N6-dimethyladenine in Streptomyces alboniger spores and culture medium.

Formation of a New Family of Aminopentose Phosphates by Extracts of Streptomyces Alboniger

The only known naturally-occurring aminopentose is 3-amino-3-deoxy-D-ribose, which has been found in three different microorganisms as part of adenine nucleosides. Fig. 1 shows the structure of two of these derivatives. 3′-Amino-3′-deoxyadenosine (Fig. 1a) was isolated from Helminthosporium sp. [2]. It is identical with adenosine except that the 3∃-hydroxyl group has been replaced by an amino group. This compound was also found in Cordyceps militaris, along with its derivative, homocitrullylaminoadenosine (Fig. 1b), which is 3′-deoxyadenosine with the amino acid, homocitrulline, linked in amide linkage to the 3′-amino group [3,4]. The first compound containing 3-aminoribose isolated from microorganisms was the antibiotic and anti-tumor agent, puromycin, whose structure is shown in Fig. 2 [5]. It is composed of a dimethyladenine moiety, a 3-aminoribose group, and an O-methyl-L-tyrosine, which here again, is attached to the amino group of ribosamine in amide linkage. No normal metabolic function in micro-organism has been discovered for any of these compounds. Puromycin, of course, has proved to be very useful to biochemists, primarily as an inhibitor of protein synthesis.