Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis.

Extracellular Penicillium fellutanum exo-beta-D-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP(2)GM(ii) and pP(25)GM(ii) (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-beta-D-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP(2)GM(ii) or pP(25)GM(ii). The k(cat)/K(m) value for pP(25)GM(ii) is 1.7 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 10(5) M(-1) s(-1), respectively. Variability in the k(cat)/K(m) values is due primarily to differences in K(m) values; the k(-1)/k(1) ratio likely provides the most influence on K(m). k(cat) increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP(x)GM(ii) added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml(-1)h(-1). No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP(x)GM(ii) and of related polymers is proportional to the k(cat)/K(m) value of each polymer. The kinetic data show that the k(cat)/K(m) value increases as the number of phosphodiesters of pP(x)GM(ii) decreases, also resulting in an increase in the activity of exo-beta-D-galactofuranosidase.

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.

Glycine betaine: reserve form of choline in Penicillium fellutanum in low-sulfate medium.

In spite of choline's importance in fungal metabolism, its sources in cytoplasm have not been fully established. 13C nuclear magnetic resonance analysis of mycelial extracts from day-5 Penicillium fellutanum cultures showed that, as well as choline-O-sulfate, intracellular glycine betaine is another reserve form of choline, depending on the availability of sulfate in the culture medium. These observations are discussed relative to the multiple roles of choline and its precursors in P. fellutanum.

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.

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum.

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.

Utilization of phosphocholine from extracellular complex polysaccharide as a source of cytoplasmic choline derivatives in Penicillium fellutanum.

Penicillium fellutanum produces a phosphorylated, choline-containing extracellular polysaccharide, peptidophosphogalactomannan (pP(x)GM) [where x is the number of phosphodiester residues]). The 13C-methyl-labeled pP(x)GM ([methyl-13C]pP(x)GM) was prepared from the cultures supplemented with L-[methyl-13C]methionine and was used as a probe to monitor the fate of phosphocholine in this polymer. The addition of [methyl-13C]pP(x)GM to growing cultures in low-phosphate medium resulted in the disappearance within 5 days of [methyl-13C]phosphocholine and N,N'-dimethylphosphoethanolamine from the added [methyl-13C]pP(x)GM. Two 13C-methyl-enriched cytoplasmic solutes, choline-O-sulfate and glycine betaine, were found in mycelial extracts, suggesting that phosphocholine-containing extracellular pP(x)GM of P. fellutanum is a precursor of intracellular choline-O-sulfate and glycine betaine. The mycelia cultured in low-phosphate (2 mM) medium contained glycine betaine and 1.5-fold more choline-O-sulfate than those grown in high-phosphate (20 mM) medium. The high levels of extracellular nonspecific phosphocholine:phosphocholine hydrolase and acid phosphomonoesterase observed in the low-phosphate culture medium are likely related to the release of phosphocholine from pP(x)GM and hydrolysis of phosphocholine, respectively. These results suggest that extracellular pP(x)GM of P. fellutanum provides phosphate needed as the environment becomes depleted of this nutrient. Choline, in excess of that needed immediately, is stored in the cytoplasm in forms that can be reutilized.

Lipid peroxidation-cytochrome P450 interactions. Use of linoleic acid hydroperoxide in the characterization of the spin-state of membrane-bound P450.

1. A procedure (linolenic acid hydroperoxide (LAHP) deletion method) is described in which LAHP is added to the reference cuvette of a pair of spectrally balanced cuvettes containing hepatic microsomes to produce a composite high spin (HS)-low spin (LS)-spectrum of P450. 2. The LAHP deletion method was used to determine the spin state of P450 in rat hepatic microsomes with and without the addition of type I compounds. 3. Advantage was taken of the temperature dependency of the spin state of P450 to determine the overall enthalpic and entropic changes for the spin equilibrium to generate computer-derived spectra of HS and LS forms of P450, and to construct a nomogram that allows direct estimation of the percentage of HS and LS spin forms of P450 in intact microsomes at temperatures compatible with biochemical functions. 4. The h.p.l.c. deletion method was used to demonstrate that HS-P450 comprised 57% of the P450 in hepatic microsomes; addition of type I substrates to these microsomes raised the level of HS-P450 to 97%. 5. The percentage of HS-P450 generated by the addition of type I compounds to microsomes declined with increasing deletions of P450 until at the extrapolated 100% level of deletion there was no HS-P450 above that of the original 57% observed in the absence of added compounds. This can be explained if LAHP destroys part of the LS-P450 while altering the remaining LS-P450 such that it retains its LS spectral characteristics but loses its capacity to form HS P450 when type I substrates are added. 6. These studies support the concept that about 50% of hepatic microsomal P450 is functionally in the HS state due to binding with high affinity endogenous substrates or other membrane components; the remaining P450 is LS-P450 that can bind to exogenous substrates to form HS-P450. 7. Applications of the LAHP deletion method for assessment of catalytic properties of membrane-bound P450 at ambient temperatures are discussed.

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.

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.

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol.

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with Km values of the order of 10(-4) M. A Km value of 60 mM was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(-)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(-)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.

Glutathathione synthetase of bovine lens: anomalies of the enzyme-catalyzed formation of ophthalmic acid.

The activity of glutathione synthetase from bovine lens was examined as a functions of the concentration of L-gamma-glutamyl-L-alpha-aminobutyrate, the dipeptide substrate required in the formation of ophthalmic acid. Several significant anomalies of the glutathione synthetase-catalyzed formation of ophthalmic acid were found. Curvilinearity of double reciprocal plots occurred with this substrate; this curvilinearity shows substrate activation of the reaction which is likely a result of negative cooperativity. Both ATP4- and, to a lesser extent Mg2+ inhibited the reaction, whereas MgATP2- is the substrate; maximum activity occurred with 2 mM Mg2+ in excess of the concentration of added ATP. This investigation shows that it is necessary to establish a defined set of conditions for reporting enzyme activity and that the usual practice of using very large concentrations of Mg2+ relative to ATP, and 5- to 20-fold excess of the dipeptide will give less than optimum activity. The unit of enzyme activity is suggested to be that activity in ml using 2 mM ATP, 4 mM Mg2+, 30 mM glycine and 15 mM L-gamma-glutamyl-alpha-aminobutyrate, which results in the formation of 1 nmole/minute of ADP or P(i). In this study, 5'-AMP was for the first time, shown to be an inhibitor of the reaction with a K(i) of 0.9 mM.

Magnitude of increase in retinal cGMP metabolic flux determined by 18O incorporation into nucleotide alpha-phosphoryls corresponds with intensity of photic stimulation.

The property of cyclic nucleotide phosphodiesterases to catalyze 3'-P--O bond cleavage and the insertion of a single nonexchangeable atom of 18O from [18O]water into the phosphoryl of the 5'-nucleotide product has been utilized as a means for measuring the hydrolytic flux of cGMP and cAMP in isolated dark-adapted intact rabbit retinas. Without illumination 18O labeling of guanine nucleotide (GTP and GDP) alpha-phosphoryls proceeds linearly for at least 80 s at a rate of 3.3 nmol of 18O/s.g of retina (wet weight). This rate is estimated to be approximately 8 times greater in the rod outer segment layer where over 90% of retinal cGMP metabolic components reside. Photic stimulation during a 20-s incubation was provided by intermittent flashes of light representing 800 ms of total illumination. Light stimuli over a range of intensities of greater than 3 log units commencing with a minimally detectable intensity produce graded increments in the rate of 18O incorporation into guanine nucleotide alpha-phosphoryls to a maximum increase of 5-fold. On the basis of only the 800-ms period of illumination this maximum increase is 125-fold. Steady state levels of retinal cGMP are not altered appreciably over this greater than 3 log range of light intensities but a light stimulus exceeding this intensity range causes an approximate 50% decrease in retinal cGMP concentration and a relative decline in the maximal rate of 18O labeling of guanine nucleotide alpha-phosphoryls. No light-related increases were detected in 18O incorporation into adenine nucleotide alpha-phosphoryls nor the gamma-phosphoryls of GTP or ATP or Pi. These observations indicate that light stimuli over greater than 3 log of light intensity produce incremental increases in cGMP metabolic flux that result from comparable increases in the rates of both cGMP generation and cGMP hydrolysis. It is postulated that increases in cGMP metabolic flux rather than changes in cGMP steady state levels are integral to phototransduction by a mechanism that involves the coupling of cGMP synthesis and/or hydrolysis to either the release of calcium from disc membranes or the inhibition of Na+ conductance by the photoreceptor membrane. This is suggested to occur by an energy-linked process and/or the generation of protons.

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.

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides.

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.

The effect of NADH and low oxygen pressure on the hepatic microsomal ethylmorphine N-demethylase activity of the male rat.

Plots of reciprocal initial rate of formaldehyde formation as a function of reciprocal ethylmorphine concentration provide parallel lines for oxygen concentrations ranging from 5.41 to 211 microM, and the apparent Km values for ethylmorphine at these extremes are 167 and 417 microM, respectively. Similarly, the apparent Km for O2 decreases from 7.69 microM at 2 mM ethylmorphine to 3.64 microM at 0.2 mM ethylmorphine. Reciprocal plots of 1/Km app and 1/Vm for ethylmorphine against 1/[O2] give Km values for O2 of 9.35 microM and 9.09 microM, respectively. Similar plots give Km values for ethylmorphine of 0.28 mM and 0.36 mM. Similarly, NADH causes an uncompetitive stimulation. These data suggest that hepatic microsomal ethylmorphine N-demethylase follows parallel plot sequential kinetics. These results, along with studies of other workers, suggest that cytochrome P-450 forms an active complex with O2 which is stable for at least many milliseconds.