Influence of osmotic and nutritional stress on physiology of Penicillium fellutanum in removal of phosphocholine and modification of phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters of peptidophosphogalactomannan species.

Addition of 3 M NaCl to 72-h cultures of Penicillium fellutanum in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP(x)GM(iii)) and a decrease in that of pP(x)GM(ii). The magnitude of (31)P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters pP(x)GM(ii) and pP(x)GM(iii) decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6-O-phospho-1'-O-[N-peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.

Primary epitopes of chicken egg yolk antibodies to peptidophosphogalactomannan.

Egg yolks from hens immunized with peptidophosphogalactomannan (pPGalMan(ii)), which contains 10 phosphocholine diester residues and is secreted by Penicillium fellutanum, contain antibodies against 5-O-beta-D-galactofuranosyl epitopes. These epitopes were the only significant determinants in pPGalMan(ii). Approximately 60-fold less pPGalMan(ii) (1.6 microM galactofuran chains) was required for 50% inhibition than galactofurano-oligosaccharides or pPGalMan containing two galactofuranosyl residues per chain.

Heat shock treatment releases Penicillium phosphoglycopeptide.

Filtrates of heat (54 degrees) treated day-5 Penicillium fellutanum cultures contained 70 mg of peptidophosphogalactomannan-II; an unheated control contained 30 mg. The polymer contained up to 60 phosphodiesters, and 5-O-beta-D-galactofuranosyl, mannopyranosyl, amino acyl and 2-aminoethanol residues. Its 13C NMR spectrum was nearly identical with that of the control polymer. The major 31P NMR signal was phosphocholine phosphodiester at delta 0.22 ppm: significant phosphodiester signals occurred at delta 1.15, 1.33 and 1.47. Dilute mineral acid released galactofuranosyl residues from the mannan. Signals at delta 1.15-1.47 ppm were associated with molecules of mass less than 3500 and contained galactose, 2-aminoethanol and peptide(s). After this acid treatment, signals at delta 1.0-0.22 remained associated with the mannan. Heat released up to 4-fold more of peptidophosphogalactomannan-III compared with the untreated control; carbohydrate and phosphate content, per mg polymer, were reduced by 4- and 2-fold, respectively. A galactofuranosyl-, phosphoryl- and amino acyl-containing polymer of Mr greater than 14,000 was solubilized by alkali treatment of P. fellutanum cell walls.

Periodate oxidation products derived from methylated alpha-amanitin: evidence for distinct aldehydic and non-aldehydic forms.

Amatoxins are cyclic octapeptides which can be purified from various mushroom species. They have found widespread use due to their potent inhibition of eukaryotic RNA polymerase II. In the course of our efforts to prepare additional semisynthetic derivatives of the amatoxin, alpha-amanitin, we examined the products formed through the periodate oxidation of 6'-O-methyl-alpha-amanitin. Periodate oxidation under conditions of near-neutral pH yielded two chemically similar, yet chromatographically separable, products. On the basis of their proton NMR spectra and their lack of reactivity with sodium chlorite these products did not contain a free aldehydic group. However, both forms were interconvertible in aqueous neutral solution and could be converted to the same product, 6'-O-methyldemethyl-gamma-amanitin, through reduction with sodium borohydride. Periodate oxidation under mildly acidic conditions generated a single product which has properties of a free aldehyde. It exhibited a proton NMR spectrum with signals characteristic of an aliphatic aldehyde and was readily oxidized to the carboxylic acid with sodium chlorite. Conditions have been defined to synthesize the free aldehyde derivative of 6'-O-methyl-alpha-amanitin in generous yield to provide a precursor for oxidation and further derivatization.

Macrocyclic pyrrolizidine alkaloids from Senecio anonymus. Separation of a complex alkaloid extract using droplet counter-current chromatography.

Ten 12-membered macrocyclic pyrrolizidine alkaloids, all of them esters of the necines, retronecine or otonecine, have been isolated from Senecio anonymus. The separation, carried out by droplet counter-current chromatography, afforded senecionine [1], integerrimine [2], retrorsine [3], senkirkine [5], neosenkirkine [6], otosenine [10], hydroxysenkirkine [7], and a new alkaloid given the trivial name anonamine [9]. Traces of usaramine [4] and another new alkaloid, hydroxyneosenkirkine [8], were detected by 1H nmr. In addition, the previously unreported 3a beta-hydroxy-4-ethoxy-2,6-perhydroindoledione [11] was isolated. X-ray structures were obtained for neosenkirkine [6], hydroxysenkirkine [7], anonamine [9], and [11]. 1H-13C heteronuclear shift correlated nmr (HETCOR) provided unambiguous chemical shift assignments for 13C-nmr data. Antitumor activity was assayed using the A204-rhabdomyosarcoma cell line in soft agarose.

Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation.

The rate at which Z. mobilis (Entner-Doudoroff pathway) converts high concentrations of glucose (20%) into ethanol plus CO2 changes as ethanol accumulates in the surrounding broth. This decline in glycolytic activity (per milligram of cell protein) does not result from inhibitory effects of ethanol, which can be reversed immediately by ethanol removal. The peak of fermentative activity (58 mumol of CO2 evolved per mg of cell protein per h) occurred after the accumulation of 1.1% ethanol (18 h) and declined to one-half this rate after 30 h (6.2% accumulated ethanol), although the cell number continued to increase. These times corresponded to the end of exponential growth and to the onset of the stationary phase (on the basis of measurement of cell protein), respectively. An examination of many of the requirements for fermentation (nucleotides, magnesium, enzyme levels, intracellular pH, delta pH) revealed three possible reasons for this early decline in activity: decreased abundance of nucleotides, a decrease in internal pH from 6.3 to 5.3, and a decrease in the specific activities of two glycolytic enzymes (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase). 31P nuclear magnetic resonance spectra of perchlorate extracts from cells fermenting in broth revealed very low levels of glycolytic intermediates (Entner-Doudoroff pathway) in cells examined at the peak of fermentative activity (18-h cells) in comparison with cells examined at a later stage (30-h cells), consistent with limitation of the fermentation rate by glycolytic enzymes near the end of the pathway. It is likely that cell death (loss of colony-forming ability) and the collapse of delta pH also contribute to the further decline in fermentative activity after 30 h.