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We recently reported that degradation of S-nitrosocysteine in homogenates of porcine aorta increased severalfold in the presence of Mg2+ ions [Kostka, P., Xu, B. & Skiles, E.H. (1999) J. Cardiovasc. Pharmacol. 33, 665-670]. The objective of the present study was to examine this in greater detail. The rate of S-nitrosocysteine degradation by aortic homogenates in the presence of Mg2+ ions exhibited differential sensitivity to chelators of iron ions. Terpyridine and diethylenetriamine penta-acetic acid (5-500 microM) caused a concentration-dependent inhibition of S-nitrosocysteine decay, whereas deferoxamine (100 microM) was ineffective. o-Phenanthroline (250 microM), a selective chelator of Fe2+ ions, potentiated the reaction at low initial concentrations of S-nitrosocysteine (< or = 15 microM) and inhibited the reaction at higher concentrations. The inhibitory effects of o-phenanthroline were related to suppression of S-nitrosocysteine decay by cysteine-mediated reduction of Fe3+. In the presence of o-phenanthroline, S-nitrosocysteine decomposition followed saturable kinetics with K0.5 = 3.8 +/- 0.3 microM and h = 1.8 +/- 0.1 (mean +/- SE, n = 4). Comparison of the rates of S-nitrosocysteine decay in different subcellular fractions showed selective association with the cytosolic fraction, as documented by copurification with lactate dehydrogenase activity. At non-limiting concentrations of S-nitrosocysteine, the rate of degradation in the cytosolic fraction was 4.1 +/- 0.3 nmol.min-1.(mg protein)-1 (n = 4). It is concluded that the cytosolic fraction of porcine aorta contains a protein factor, presumably an enzyme, capable of catalyzing heterolytic decomposition of the S-NO bond of S-nitrosocysteine in a process involving redox cycling of iron ions.

In plant, the catabolism of lysine has only been studied in some detail in maize. The enzymes lysine 2-oxoglutarate reductase (also known as lysine alpha-ketoglutarate reductase; LOR) and saccharopine dehydrogenase (SDH), which convert lysine into saccharopine, and saccharopine into glutamic acid and 2-aminoadipate 6-semialdehyde, respectively, were isolated from immature rice seeds and partially purified through a three-step purification procedure involving ammonium sulphate precipitation, and anion-exchange and gel-filtration chromatographies, leading to a final yield of 30% for LOR and 24% for SDH. The molecular masses estimated by gel-filtration chromatography on a Sephacryl S200 column and by native non-denaturing PAGE using Ferguson plots were 203 kDa for both enzymes by gel-filtration and 202 kDa for both enzymes by native non-denaturing PAGE. A second band of LOR and SDH activities on native gels was observed for both enzymes with an estimated molecular mass of 396 kDa, which indicated a multimeric structure. Kinetic studies were consistent with an ordered sequence mechanism for LOR, where 2-oxoglutarate is the first substrate and saccharopine is the last product. The results observed for the LOR/SDH activity ratios during purification, the copurification in all three steps, the molecular masses, the relative mobilities on native non-denaturing gels and the pI estimated for LOR and SDH suggest the existence of a bifunctional polypeptide containing LOR and SDH activities.

The use of glutathione reductase inhibitors in chemotherapy is the raison d'être for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N-terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first beta strand and the diphosphate-fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P.falciparum glutathione reductase as a drug target are discussed.

The purification by affinity chromatography of beta-galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L-serine deaminase 1. We conclude that sdaA is the structural gene for the latter enzyme. The purified L-serine deaminase 1 obtained after collagenase treatment of an sdaA-collagen-lacZ fusion differs from the native enzyme by the addition of several amino acids at the C-terminal. Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.

The oligodendrocyte-specific UDP-galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte- and myelin-specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton-X-100-solubilized rat brain microsomes by ion exchange, dye ligand and lectin affinity chromatography as a 64-kDa protein homogenous in SDS/PAGE. It copurified with the brain-specific Na(+)-dependent high-affinity L-glutamate/aspartate neurotransmitter transporter (GLAST-1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high-mannose glycoprotein and GLAST-1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70-kDa isoform of GLAST-1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70-kDa isoform differs only in the degree of glycosylation from the 64-kDa GLAST-1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein-chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST-1 were successfully applied to the cloning of CGT and the first high-affinity glutamate neurotransmitter transporter (GLAST-1) in glia of the central nervous system as well.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.

About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) cells, which is consistent with the view that actin is maintained in different pools (F-actin--ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 degrees C of 0.1 +/- 0.025 s-1 by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.

A procedure has been worked out which allowed the purification and crystallization of a citrate lyase/citrate lyase ligase complex from Rhodopseudomonas gelatinosa. The complex was subsequently separated to yield two homogeneous enzymes. Citrate lyase ligase was purified 365-fold with a yield of 3.23%. The molecular weight of the enzyme was estimated to be 39500, the enzyme consisted of one polypeptide chain. The reaction rates for ATP, acetate and citrate lyase (sulfhydryl form) followed Michaelis-Menten kinetics (Km values: 0.14 mM, 5 mM and 37 nM respectively). Citrate lyase ligase exhibited a high substrate specificity and could not react with citrate lyases from nonphototrophic microorganisms. In contrast to the ligase from Streptococcus diacetilactis, the enzyme from R. gelatinosa was extremely labile; however, it could be stabilized by nucleotides, the most potent stabilizing one being ADP.