Constantinos G. Zarkadas

Formyltetrahydrofolate dehydrogenase-hydrolase from pig liver: simultaneous assay of the activities.

The bifunctional folate-dependent enzyme, 10-formyltetrahydrofolate dehydrogenase-hydrolase (10-formyltetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.6), has been purified to homogeneity from pig liver. Its amino acid composition was determined and gave a calculated v of 0.735 ml/g; a molecular weight of 92500 for the protein subunit was determined as well. Spectrophotometric, fluorescence emission and radiochemical methods were devised to assay the activities. Quantitative separation of carbon dioxide and formate produced by the dehydrogenase and the hydrolase reactions, respectively, demonstrated that both activities occur simultaneously. This fact, together with a 5-fold difference in the Km values for the folate substrate, strongly suggests that these two activities are functions of different sites. The possible role of polyglutamate specificity for the preferential selection of one of the activities under physiological conditions was ruled out when both proved to have similar specificities, as determined by sensitivity to inhibition by tetrahydropteroylpolyglutamates.

Rapid method for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine and the amino sugars in proteins and tissues.

A rapid and sensitive chromatographic method is described for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine, glucosamine and galactosamine at picomole levels in protein and tissue hydrolysates. This method uses either an automated amino acid analyser with a 17.5 X 0.28 cm microcolumn packed with 6.0 +/- 0.5 micron spherical resin, thermostated at 52 degrees C, one buffer system (0.21 M sodium citrate, pH 5.125) and 3-nitrotyrosine as the internal standard, or conventional instruments using the same system but with larger diameter columns and resins (11.0 +/- 1.0 micron). This method should be especially valuable for determining collagen and elastin in tissue hydrolysates from the amounts of 5-hydroxylysine, and desmosine or isodesmosine present, respectively, and for studying protein hydroxylation, glycosylation, cross-linking formation, and the turnover rates of collagen and elastin in normal and diseased tissues.

Determination of methylated basic, 5-hydroxylysine, elastin crosslinks, other amino acids, and the amino sugars in proteins and tissues☆☆☆

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.

The nitrogenase proteins of Rhizobium meliloti: purification and properties of the MoFe and Fe components

The alfalfa-Rhizobium meliloti symbiosis contributes a major portion of biologically fixed nitrogen to temperate zone forage crop production. Highly-purified molybdenum-iron (MoFe) and iron (Fe) nitrogenase components were obtained for the first time from extracts of R. meliloti bacteroids. Intact bacteroid cells were isolated anaerobically from 100 g quantities of alfalfa nodules following storage in liquid nitrogen. Centrifuged bacteroid extracts showed a marked reduction in specific activity when assayed at protein concentrations less than 1 mg/ml. Both nitrogenase proteins were resolved and purified to homogeneity as determined spectroscopically and by SDS-PAGE. The purified MoFe protein differed in several respects from previously characterized nitrogenase proteins. Saturation of the acetylene-reducing and proton-reducing activities of the R. meliloti MoFe protein required higher relative concentrations of Fe protein than nitrogenase proteins purified from free living diazotrophs. Electron allocation to dinitrogen reduction was sustained at component ratios similar to those present in bacteroid extracts, suggesting that while the observed saturation effects were not detrimental to physiological function in the symbiotic system, overall activity could be enhanced by higher levels of iron protein. Analyses of the MoFe protein gave 22 Fe, 22 labile sulfide and 1.7 Mo atoms per molecular unit of 215 kDa. Dithionite-reduced MoFe protein contained a spin 3/2 iron centre but had a lower visible absorbance at 360 nm than the equivalent Azotobacter chroococcum component. Amino-acid composition indicated a notably lesser tryptophan content, and cysteine content greater than that of the equivalent tetrameric protein of free living diazotrophs. Ratios of acidic and basic residues were similar to other MoFe proteins. Calculation of hydrophobicity and discriminant parameters gave values midway between those expected for soluble cytoplasmic proteins and peripheral membrane associated proteins. ADP was tightly bound by the dithionite-free MoFe protein containing reduced iron-molybdenum cofactor. The R. meliloti iron protein was found to be a 64 kDa homodimer containing a single 4Fe-4S metal centre.

Different sites for fatty acid activation and acyl transfer by the synthetase subunit of fatty acid reductase: acylation of a cysteinyl residue

Abstract Fatty acid reductase from the luminescent bacterium Photobacterium phosphoreum catalyzes the NADPH- and ATP-dependent conversion of fatty acid into the corresponding aldehyde required for light emission. The mechanism involves acyl-AMP formation, followed by acylation of the synthetase subunit, acyl transfer to the reductase subunit and reduction with NADPH. Modification with N-ethylmaleimide of a highly reactive thiol in the acylprotein synthetase subunit prevents acylation but not acyl-AMP formation, indicating that these two steps take place at different sites in the synthetase subunit. Prior acylation of the enzyme, but not binding of fatty acid and ATP, protects against inactivation by N-ethylmaleimide. Treatment of the acylated enzyme with neutral hydroxylamine results in deacylation. Moreover, the proteolytic peptide maps of the synthetase labelled with N-[ 3 H ] ethylmaleimide and with [3H]tetradecanoic acid appear to be identical. These results indicate that acylation takes place at a specific cysteine residue in the synthetase subunit. Interaction with the reductase subunit, which stimulates the acylation of the synthetase subunit, partially protects the acylation site against modification with N-ethylmaleimide. Aminoacid analysis gave molecular weights of 34 000, 52 000 and 57 000 for the transferase, synthetase and reductase polypeptides, respectively, based on best fit integral analyses, in good agreement with molecular weights determined by SDS gel electrophoresis. The average hydrophobicity and the content of polar residues indicate that the synthetase subunit as well as the transferase subunit is less hydrophobic than the reductase subunit, in agreement with the isolation of the two former subunits as monomers and the latter subunit as a tetramer on resolution from the fatty acid reductase complex.

Seasonal variation of hydrophilic, hydrophobic, and charged amino acids in developing apple flower buds 1

Abstract An increase in polar amino acids especially in hydrophilic and charged amino acids and a decrease in hydrophobic amino acids were observed in developing apple flower buds from July to November (beginning of dormancy). This suggests that polar amino acids may play a role in dormancy of apple flower buds similar to that of other polar substances. A 2nd‐degree polynomial was observed for hydrophilic, hydrophobic, and charged amino acid content, during growth and development.

An improved procedure for the purification of formiminotransferase-cyclodeaminase from pig liver. Kinetics of the transferase activity with tetrahydropteroylpolyglutamates.

Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.

Determination of methylated basic amino acids, 5-hydroxylysine, and elastin crosslinks in proteins and tissues

Myosin, actin, collagen, and elastin of adult bovine diaphragm and porcine and avian skeletal muscle tissues and their separated intracellular and extracellular muscle protein fractions have been determined by chromatographic methods developed to quanti tate their unique basic amino acids. In this direct chemical approach the determination of the myofibrillar myosin and actin content of skeletal muscle is based on the amounts of Nτ-methylhistidine present, and collagen from the amount of 5-hydroxylysine present. Elastin was also determined from the amounts of desmosine found. Skeletal muscles were found to contain between 10.2–11.5% actin and 21.3 to 24.0% myosin, corresponding to 21.1% and 44.0% of the myofibrillar proteins (52.3% of the total muscle proteins), between 37.6–46.3% other SDS-soluble intracellular proteins, and 4.7 to 10.2% SDS-insoluble extracellular matrix muscle proteins. Total skeletal muscle collagan ranged from 2.8 to 5.9%, while elastin accounted for an estimated 0.063–0.143%, and the transcellular matrix SDS-insoluble proteins accounted for the remaining 1.8%.