In vivo biosynthesis of peptidophosphogalactomannan in Penicillium charlesii.

The major exocellular glycopeptide (peptidophosphogalactomannan) produced by Penicillum charlesii first appears in the culture filtrate when the growth medium is nearly depleted of NH4+. The extent of incorporation of exogenously supplied radioactive precursors (D-[U-14C] GLUCOSE, L-[U-14C]threonine and NaH2(32)PO4) into peptidophosphogalactomannan suggests that approximately 20% of the total quantity of peptidophosphogalactomannan is assembled from constituents taken from the growth medium before NH4+ starvation and that the remainder is assembled from constituents in the medium during NH4" starvation. In the absence of NH4+, an increase in dry weight continues until the medium is depleted of glucose. However, peptidophosphogalactomannan accumulation proceeds until after glucose is depleted and growth is halted. These data suggest that peptidophosphogalactomannan is a product of cellular turnover.     

Influence of 2-deoxy-D-glucose on galactofuranosidase and peptidophosphogalactomannan synthesis in Penicillium charlesii

2-deoxy-D-glucose inhibits synthesis of the glyco-enzyme $\text{exo}$-β-D-galactofuranosidase and its secretion into the growth medium of $\text{Penicillium charlesii}$ cultures. In contrast, the synthesis of peptidophosphogalactomannan, an extracellular glycopeptide (peptido-polysaccharide) occurs in nearly normal quantities in the presence of 2-deoxy-D-glucose. The peptidophosphogalactomannan's composition in cultures containing 2-deoxy-D-glucose was comparable to that obtained from cultures containing no added 2-deoxy-D-glucose. We conclude peptidophosphogalactomannan is not derived from mural or extracellular glycoprotein(s) whose synthesis is inhibited by 2-deoxy-D-glucose.     

The 5-O-beta-D-galactofuranosyl-containing peptidophosphogalactomannan of Penicillium charlesii. Characterization of the mannan by 13C NMR spectroscopy

Penicillium charlesii secretes a galactofuranosyl and phosphodiester-containing peptidophosphogalactomannan (pPGM). A linear mannan was prepared from pPGM by treatment with 48% aqueous HF which selectively cleaves galactofuranosyl and phosphodiesters; treatment with alkaline borohydride releases the mannan from the polypeptide. Mannan from P. charlesii cultured in D-[1,2-13C2]glucose contained mannopyranosyl residues which were enriched in 13C at both C-1 and C-2 and, to a lesser extent, at C-5 and C-6. The mannan was examined with a combination of 13C NMR INADEQUATE pulse sequence and selective 13C saturation to assign the resonance frequency of anomeric carbons directly coupled to specific C-2 signals. Three species of mannosyl residues, each substituted with a glycosidic linkage at C-2, and a fourth species substituted at C-6 and not substituted at C-2 were observed. Mannan obtained from P. charlesii cultured in D-[6-13C]glucose contained mannopyranosyl residues which were enriched in 13C primarily in C-6. The mannan was examined by DEPT 13C NMR to determine the number of species which were substituted at C-6. Mannan, treated as described above, contained a 1----6-linked mannopyranosyl species. pPGM contains minor quantities of at least four other substances attached to hydroxymethyl groups of the hexosyl residues.     

Galactofuranosyl-containing glycopeptide of Penicillium charlesii. Vacuum ultraviolet circular dichroism

The far and vacuum u.v. circular dichroism (CD) of peptidophosphogalactomannan from P. charlesii is reported to 182.5 nm in aqueous and aqueous/organic solvents, and to 150nm in films. CD of films of the peptide-free derivative is reported to 150 nm. On the basis of these data we conclude that the peptide chain is unordered, and may best be described as a hydrated coil showing some stiffness. The small observed saccharide CD may result from cancellation of contributions from the various saccharide structures present or from a lack of repeating secondary structure.     

Isolation of Citreoviridin from Penicillium charlesii Cultures and Molded Pecan Fragments

The extraction and systematic fractionation of Penicillium charlesii Smith cultures and contaminated pecan fragments yielded the yellow mycotoxin citreoviridin. Citreoviridin proved acutely toxic to 1-day-old chickens, with an oral 50% lethal dose of 37.5 mg/kg, and showed plant growth inhibition in wheat coleoptiles even at concentrations as low as 10⁻⁴ M. It was toxic to corn seedlings but did not affect young tobacco seedlings.     

Isolation, purification, and properties of Penicillium charlesii alkaline protease.

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Exocellular glycopeptide from a Penicillium charlesii mutant incapable of growth on D-galactose.

The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by O-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1 yields 5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1 yields 2)-linked (67.5%) and (1 yields 6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1 yields 6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.     

In vivo biosynthesis of cholecystokinin in hog cerebral cortex

The biosynthesis of cholecystokinin (CCK) in the cerebral cortex of hogs was studied by intracisternal injections of [³⁵S]methionine. At different times (15, 60 and 120 min) after the injection, cortex was isolated and extracted with boiling water and 0.5 M acetic acid. CCK in the extracts was immunosorbed, using an antiserum specific for the COOH-terminal sequence of CCK. Subsequently, the CCK-immunoreactivity was applied to Sephadex G-50 superfine columns. The fractionation showed incorporation in five molecular forms with elution constants of 0.08, 0.50, 0.90, 1.1 and 1.3. After a pulse period of 15 min, [³⁵S]methionine was incoporated mainly into the largest form of CCK (K_av of 0.08). The incorporation in all forms increased during the first hour. After 2 hours, a decline occurred in the larger forms, whereas the incorporation in the octapeptide form and tetrapeptide-like form increased.     

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

Studies on the biochemistry of Penicillium charlesii. Influence of various dicarboxylic acids on galactocarolose synthesis

1. It has been shown that Penicillium charlesii continues to synthesize galactocarolose when l-malic acid, malonic acid, succinic acid, fumaric acid, maleic acid or oxaloglycollic acid is substituted for dl-tartaric acid in the Raulin-Thom nutrient medium. 2. The quantity of galactocarolose synthesized per g. of mycelia was markedly decreased by substitution of l-malic acid, malonic acid, succinic acid, fumaric acid or maleic acid for dl-tartaric acid. Substitution of oxaloglycollic acid for dl-tartaric acid did not depress the galactocarolose synthesized/g. of mycelia; however, the quantity of fungal mass formed was decreased approximately fivefold. 3. Based upon (14)C incorporation into galactocarolose, succinic acid, fumaric acid or malonic acid did not serve as direct precursors of galactose as did tartaric acid. Oxaloglycollic acid, l-malic acid and maleic acid were not tested. 4. The relative quantity of galactocarolose synthesized per g. of mycelia decreased as the concentration of diammonium dicarboxylate added to the growth medium was increased. Tartaric acid, oxaloglycollic acid, fumaric acid and malonic acid were tested. 5. The quantity of mycelia formed and the quantity of galactocarolose synthesized per g. of mycelia were greater when the growth medium contained l-tartrate than when it contained d-tartrate.     

In vivo biosynthesis of vacuolar proteinases in proteinase mutants of Saccharomyces cerevisiae

The molecular forms of proteinase A, proteinase B and Car?ypeptidase Y, enzymes of the lysosome like yeast vacuole, were studied in mutants (Wolf,D.H. and Fink,G.R. (1975) J. Bacteriol. 123, 1150–1156;Wolf,D.H. and Ehmann,C. (1978) FEBS Lett. 92, 121–124;Mechler,B. and Wolf,D.H. (1981) Eur. J. Biochem. 121, 47–52) defective in genes, which appear to be structural genes of the respective enzymes. According to the immunochemical reactivity of proteinase protein, mutants could be divided into three classes: 1) Mutants harboring no immunoreactive proteinase material. 2) Mutants synthesizing proteinase precursor molecules of similar size as wild type, which are transferred into mature proteins, which are, however, completely inactive. 3) Mutants synthesizing proteinase precursor-like proteins, which are not processed into the mature proteins. As measured in the car?y-peptidase Y mutant strain the mutant precursor car?ypeptidase Y is rapidly degraded. A pleiotropic mutation (pep4-3) resulting in low activities of five vacuolar enzymes had been shown to accumulate pro-car?ypeptidase Y like immunoreactive material (Hemmings,B.A., Zubenko,G.S., Hasilik,A., and Jones,E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435–439). We found that this mutant is also defective in processing the proteinase B precursor, whereas no cross-reactive proteinase A molecule could be detected under the conditions employed.