Interaction of 4-aminobutyrate-transaminase from swine kidneys with 5'- and 6'-methyl derivatives of pyridoxal-5'-phosphate

The study of interaction of 4-aminobutyrate transaminase with 5'- 6'-methyl derivates of PLP demonstrated that only the former was capable of forming a catalytically active holoenzyme possessing 0.37 activity of the native holoenzyme and a low affinity substrates. This compound interacts with the apoenzyme at a slower rate than does PLP; it has a reduced affinity towards apotransaminase (Km = 1.10(-4) M) and is replaced from the active site by native coenzyme. The other analog of pyridoxal-5'-phosphate forms a catalytically inactive complex with the apoenzyme; the other analog is not replaced from the active center by native coenzyme and non-competitively inhibits the reconstruction of apotransaminase (Ki = 2.10(-5) M).     

Effect of postweanling pyridoxine deficiency on growth and concentration of the coenzyme pyridoxal-5'-phosphate in heart, kidneys, lungs, and adrenals in rats

Dietary pyridoxine deficiency induced in postweanling rats to led to severe growth retardation and growth failure, with a sharp increase in the mortality rate after about 8 weeks on the diet. The absolute weights of heart, lungs, kidneys, and adrenals were all lower in the deficient animals than those of the controls. However, when the weights were expressed in terms of percentage of body weight, a 2-fold increase was observed indicative of marked hypertrophy. The depletion of the coenzyme pyridoxal-5'-phosphate from these tissues was extensive (heart 48%, lungs 85%, and kidneys and adrenals 88%.) The extent of loss of the coenzyme from the tissues suggests possible functional changes.     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

Inhibition of replication of rinderpest virus by 5-fluorouracil

Groups of male Wistar rats lived in cages capable of monitoring feeding and drinking continuously at 0.1-s intervals, 24 h per day. Intact animals were subjected to 20 min of restraint stress or to brief handling (Brief Pick-Up), daily or every third day, 6 h after the beginning of the 12-h light period. In both studies, food-intake increased in the first hour after restraint, peaking between 15 and 45 min. Smaller increases were seen following Brief Pick-Up. More interestingly, the amount of food eaten increased across test sessions, indicating sensitization of the response to stress. Drinking also increased following stress, occurring before feeding and diminishing after the first 15 min. In adrenalectomized animals implanted with slow-release pellets to replace basal diurnal levels of corticosterone (ADX animals), sensitization of the feeding response to restraint stress developed across test sessions, although in these animals, the acute increase in food-intake following restraint stress was attenuated. ADX animals subjected only to Brief Pick-Up showed no increases in food-intake. Daily injections of 3.0 mg/kg corticosterone given to such ADX animals were unable to mimic the effects of restraint on either food-intake or drinking, nor did they augment the effects of restraint in ADX animals. We conclude that sensitization to the effects of brief restraint stress on food-intake can occur independently of a stress-induced rise in plasma corticosterone.     

On the mobility of riboflavin 5'-phosphate in Megasphaera elsdenii flavodoxin as studied by 13C-nuclear-magnetic-resonance relaxation

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (greater than 90% 13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP. In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.     

Pyridoxal 5'-phosphate and the regulation of ornithine decarboxylase activity and stability

There are two forms of ornithine decarboxylase with respect to pyridoxal 5'-phosphate (pyridoxal-P) affinity in exponentially-growing Swiss 3T3 mouse fibroblasts: form I (Km approximately 10 muM) accounts for 30% of the total activity, and form II (Km approximately 0.4 muM) the remainder. Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal-P; neither form recognizes the Schiff base between ornithine and pyridoxal-P as a substrate. Total pyridoxal-P concentrations indicate that both forms may normally be at least partially active in vivo. Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10-fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme. The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association. The half-lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half-life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal-P serving a major protective function for the enzyme in vivo.     

Abnormal regulation of plasma pyridoxal 5'-phosphate in patients with liver disease

Pyridoxal 5'-phosphate (PLP), the coenzyme form of vitamin B6, is essential for many biochemical reactions in the body. Studies in experimental animals have suggested that the liver is a primary site for the formation of PLP circulating in the plasma, and that it may also participate in its degradation. This study evaluates, for the first time, the effects of liver disease in man on the regulation of plasma PLP. The plasma PLP level was measured before and sequentially after the rapid intravenous administration of 50 mg of pyridoxine to patients with alcoholic cirrhosis, acute hepatitis, and extrahepatic obstruction, and to normal control subjects. The base line plasma PLP concentration was significantly lower in cirrhotic patients than in normal persons (P less than 0.025), and there was a tendency for it to be reduced in patients with extrahepatic obstruction. After administration of pyridoxine there was a significant increase in the plasma PLP level over a 2- to 12-hr period, after which the concentration returned gradually toward the initial value. The area under the concentration/time curve was from 2 to 8 times smaller (P less than 0.002) in the patients with liver disease. To assess possible mechanisms of this change, 5 mg of PLP were intravenously administered to the various patient groups and the pharmacokinetics of the disposition were assessed. The initial and steady state volumes of distribution of PLP were comparable in cirrhotics and controls (P greater than 0.05), but the clearance of plasma PLP in cirrhotics was much faster (63.0 +/- 7.4 versus 31.7 +/- 2.7 ml per min, P less than 0.004). Similar findings were obtained in the other liver disease subjects. The in vitro plasma binding of PLP at supracirculatory concentrations was comparable in cirrhotics and controls (99.4 versus 99.5%, P greater than 0.05). In conclusion: (1) plasma PLP regulation in patients with liver disease is abnormal, (2) a significant factor in the decrease in plasma PLP after intravenous pyridoxine administration in these patients appears to be an increase in the total plasma clearance of the coenzyme, and (3) it is postulated that this may be due to increased degradation of PLP by the diseased liver.     

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil

Interest in the health-promoting effects of virgin olive oil, an important part of the 'Mediterranean diet', prompted us to determine the anti-eicosanoid and antioxidant effects in leukocytes of the principal phenolic compounds from the 'polar fraction': oleuropein, tyrosol, hydroxytyrosol, and caffeic acid. In intact rat peritoneal leukocytes stimulated with calcium ionophore, all four phenolics inhibited leukotriene B4 generation at the 5-lipoxygenase level with effectiveness hydroxytyrosol > oleuropein > caffeic acid > tyrosol (approximate EC50 values: 15, 80, 200, and 500 microM, respectively). In contrast, none of these compounds caused substantial inhibition of thromboxane generation via the cyclo-oxygenase pathway. Hydroxytyrosol, caffeic acid, oleuropein, and tyrosol (decreasing order of effectiveness) also quenched the chemiluminescence signal due to reactive oxygen species generated by phorbol myristate acetate-stimulated rat leukocytes. None of these compounds were toxic to leukocytes at the concentrations tested. We conclude that the phenolics found in virgin olive oil possess an array of potentially beneficial lipoxygenase-inhibitory, prostaglandin-sparing, and antioxidant properties.     

Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate

Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.     

Ornithine decarboxylase from Leishmania mexicana promastigotes: interaction with pyridoxal 5'-phosphate and alpha-difluoromethylornithine

The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.     

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.     

Light-dependent activation of rod transducin by pineal opsin

The pineal gland expresses a unique member of the opsin family (P-opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502-1506) that may play a role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope-tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a lambdamax at 462 +/- 2 nm. P-opsin bleached slowly in the dark (t1/2 = 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTPgammaS bound/mol of P-opsin/min at 7 microM) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.