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Alpha-amanitin, a toxin from the basidiomycete, Amanita phalloides, has been shown to specifically inhibit the DEAE fraction II enzyme (2-5) that is found in the nonnucleolar portion of the nucleus. It has been suggested that the fraction II enzyme functions in the synthesis of DNA-like RNA. The fraction I enzyme's cellular location is in the nucleolus, and it is reported to serve specifically in the synthesis of ribosomal RNA (1, 2, 6). At present there is no evidence or suggested function for the fraction III enzyme, which is also apparently extra-nucleolar (5, 6). Although it has been suggested that the polymerases have different cellular functions, for more conclusive investigations specific inhibitors would be very useful.While it is generally accepted that cycloheximide interrupts protein synthesis in eukaryotes, there have also been reports that it has some effect on the synthesis of RNA (7-10). The antibiotic rifampicin and rifamycins in general have been shown to specifically inhibit procaryotic RNA polymerases (11-13).The purpose of this study was to examine the RNA synthesis machinery in the primitive water mold Blastocladiella emersonii. Since the results indicated the presence of multiple RNA polymerases, another objective was to observe the effects of cycloheximide, alpha-amanitin, and rifampicin on in vitro RNA synthesis to determine whether specific inhibitors may exist for the three polymerases.MATERIALS AND METHODS Growth, harvesting, and disruption of cells Single-generation cultures of B. emersonii (Strain 49-1) were grown in aerated carboys at 230C according to Horgen and Griffin (14). Vegetative, ordinary colorless thalli were harvested by filtration and were washed with glass distilled water. The thalli were suspended in homogenizing fluid (15) and were disrupted with a French pressure cell. The cultures and the enzyme preparations were monitored for microbial contamination by plating on peptone-yeast extract-glucose agar (PYG) with negative results. ChemicalsMedium components were purchased from Difco Laboratories, Detroit, Mich. The unlabeled nucleoside triphosphates and dithiothreitol were purchased from Sigma Chemical Co., St. Louis. The [3H]ATP was obtained from New England Nuclear Corp., Boston. Salmon sperm DNA and enzyme grade ammonium sulfate were obtained from Mann Research, N.Y. Alpha-amanitin was purchased from Henley and Co., N.Y., rifampicin and cycloheximide (actidione) from Calbiochem, Los Angeles. Other chemicals used were reagent grade.Isolation of crude nuclear pellet Cell wall debris was removed by filtration through Miracloth and the homogenate was centrifuged for 20 min at 6000 X g.The resulting crude nuclear pellet was used for enzyme isolation. Further nuclear purification by centrifugation through 4 molal sucrose (15) did not increase the purity of the enzyme preparations.Purification of RNA polymerase RNA polymerase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose column chromatography after Mertelsmann and Matthaei (16)...

The enzymatic conversion of xanthoxin to abscisic acid by cellfree extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.

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The inhibition of indoleacetic acid oxidase activity by chlorogenic acid is markedly variable. Aqueous extracts of acetone precipitates from etiolated pea epicotyls contain heat-stable substances which decrease the inhibition produced by chlorogenic acid and also have a small activating effect. Low concentrations of pcoumaric acid (1 micro/ml) can stimulate this action. The possible role of activators and inhibitors in the in vivo regulation of indoleacetic acid oxidase activity is discussed.