Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

The control of plant glutamate dehydrogenase by pyridoxal-5′-phosphate

The proposition that the nitrogen status of a plant is reflected by the ratio pyridoxal phosphate to pyridoxamine phosphate and that this ratio exerts a controlling influence on plant metabolism has been examined. The ratio pyridoxal phosphate to pyridoxamine phosphate has been shown to increase during nitrogen starvation. The inhibition of glutamate dehydrogenase by pyridoxal phosphate has been examined and the kinetics of inhibition are discussed in relation to the proposed control of metabolism.     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Modification and identification of glutamate residues at the arginine-recognition site in the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

It has been proposed that the active centre of cyclic AMP-dependent protein kinase contains an arginine-recognition site, which is considered to be essential for the function of the catalytic subunit of the kinase [Matsuo, Huang & Huang (1978) Biochem. J.173, 441-447]. The catalytic subunit can be inactivated by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide and glycine ethyl ester at pH6.5. The enzyme can be protected from inactivation by preincubation with histone, a protein substrate of the enzyme. On the other hand, ATP, which also serves as a protein kinase substrate, does not afford protection. Polyarginine, a competitive inhibitor of protein kinase, which is known from kinetic studies to interact specifically with the arginine-recognition site, partially protects the catalytic subunit from inactivation by 3-(3-dimethylaminopropyl)-1-ethylcarbodi-imide. These results lead to the conclusion that the site of modification by carbodi-imide/glycine ethyl ester is most likely located at the arginine-recognition site of the active centre. A value of 1.7+/-0.2 (mean+/-s.d.) mol of carboxy groups per mol of catalytic subunit has been obtained for the number of essential carboxy groups for the function of protein kinase; a complete chemical modification of these essential carboxy groups results in total loss of catalytic activity. Finally, we have identified the essential carboxy group in the catalytic subunit of cyclic AMP-dependent protein kinase as being derived from glutamate residues. This is achieved by a three-step procedure involving an extensive proteolytic digestion of the [1-(14)C]glycine ethyl ester-modified enzyme and two successive high-voltage electrophoreses of the hydrolysate. It is concluded that 1.7mol of glutamyl carboxy groups per mol of catalytic subunit may be considered a component of the arginine-recognition site in the active centre of cyclic AMP-dependent protein kinase.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

Affinity Labeling of the Guanine Nucleotide Binding Site of Transducin by Pyridoxal 5′-Phosphate

Transducin (T), a guanine nucleotide binding regulatory protein composed of α-, β-, and γ-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5′-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [³H]NaBH₄. Approximately 1 mol of ³H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of ³H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the α- and β-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.     

Stimulation of ion transport by ascorbic acid through inhibition of 3′:5′-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues

Ascorbic acid stimulates active transport of Cl^? by the isolated intact corneas. The effect is not present in corneas previously stimulated by theophylline, an inhibitor of 3′: 5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3′: 5′-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using ³H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46 %. This compares with 58 % inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45 % is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50 %) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12 %, rat brain (caudate nucleus) 48 %, rabbit brain 14 %, rabbit liver 16 %. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3′: 5′-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

The identification of the site of action of NN′-dicyclohexylcarbodi-imide as a proteolipid in mitochondrial membranes

Mitochondrial membranes were incubated with NN'-dicyclohexyl[(14)C]carbodi-imide, which irreversibly inhibited the partial reactions of oxidative phosphorylation by 95-100%. Solutions of the membranes were analysed on polyacrylamide gels. Of the radioactivity recovered from the gels 90% was shown to be associated with a single protein of molecular weight about 10000. The radioactive protein and associated phospholipid was solubilized from the membrane by extraction with chloroform-methanol mixtures and was concentrated 50-fold by solvent fractionation and adsorption chromatography on Sephadex LH-20. Several protein-radioactivity peaks were obtained by Sephadex LH-20 chromatography. However, 90-100% of the radioactivity in each peak was shown to be associated with a single protein similar to the major radioactive protein observed in electrophoretograms of the membrane solutions. It is concluded that dicyclohexylcarbodi-imide inhibits mitochondrial oxidative phosphorylation by reacting covalently with a group on this chloroform-methanol-soluble protein. The possible role of this protein in oxidative phosphorylation is discussed.     

The peptidyltransferase center of Escherichia coli ribosomes: Binding sites for the cytidine 3′-phosphate residues of the aminoacyl-tRNA 3′-terminus and the interrelationships between the acceptor and donor sites

The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70 S ribosomes was investigated in Ac-Phe-tRNA·poly(U)·70 S ribosome (system A) and tRNA^Phe·poly(U)·C-A-C-C-A-Phe·70 S ribosome (system B) systems by using C-C-A-Gly, C-C-A-Phe, C-A-Gly and C-A-Phe as analogs of the 3′-terminus of aminoacyl-tRNA. It was found that an addition of C_p residue to C-A-Gly and C-A-Phe resulted in an increase of the acceptor activity in system A; the increase is more remarkable for C-A-Gly than for C-A-Phe, while the acceptor activities of C-C-A-Gly and C-C-A-Phe are roughly similar. On the other hand, dramatically increased binding affinities of C-C-A-Phe and C-C-A-Gly relative to C-A-Phe and C-A-Gly for the A site of peptidyltransferase were observed in system B using an inhibition assay; C-C-A-Phe binds much more strongly than C-C-A-Gly. The results indicate the important role of the third C_p residue and the aminoacyl moiety of the 3′-terminus of aminoacyl-tRNA in the interaction with the acceptor site of peptidyltransferase, as well as the existence of cooperative effects between A and P sites of peptidyltransferase. These effects, depending on an occupancy of P site, may significantly influence the specificity of the peptidyltransferase A site.     

Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine

Summary The accessibility of pyridoxal 5′-phosphates of the phosphorylaseab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of theb anda forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylasea andab to resolution at 0°C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of theab hybrid was similar to that of thea and differed from that of theb forms in that it occurred at 30°C and 37°C but not at 0°C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of theab form between that of theb anda forms. We conclude that partial phosphorylation of phosphorylaseb elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5′-phosphates. Resistance ofab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylaseb anda.     

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

The use of the GDH gene for molecular identification and phylogenetic analysis of the yeast Kluyveromyces marxianus

Summary Our previous work showed that NADP⁺-dependent glutamate dehydrogenase from K. marxianus behaves similarly to its counterpart in S. cerevisiae. It suggested that the ammonia assimilation pathway might be different between K. marxianus and the genetic closed species K. lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K. marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K. marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K. marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S. cerevisiae GDH1 gene family together with KlGDH gene.     

The anti-fibrotic effect of betulinic acid is mediated through the inhibition of NF-κB nuclear protein translocation

The purpose of the study was to investigate the anti-fibrotic effect and the potential mechanisms of action of betulinic acid (BA) against hepatic fibrosis in vivo and in vitro. BA is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae). Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200mg/kg) twice weekly for 6weeks in Wistar rats. The administration of BA (20 or 50mg/kg) was started following TAA injections and was continued for 6 or 8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver tissue hydroxyproline and α-smooth muscle actin (α-SMA). In vitro, BA effectively decreased the HSC-T6 cell viability induced by TNF-α and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of α-SMA and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner. This study provides evidence that BA exerts a significant anti-fibrosis effect by modulating the TLR4/MyD88/NF-κB signaling pathway.     

A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1

Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.     

Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH₄. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH₄. One mole of N-?-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.     

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by α1-antitrypsin

The role of lysines 37–39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and α₁-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by α₁-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with α₁-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI–heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI–APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by α₁-antitrypsin, two important blood plasma serpins.