An exo-D-galacturonanase of Butyrivibrio fibrisolvens from the bovine rumen

An intracellular pectinolytic enzyme was isolated from a cell extract of Butyrivibrio fibrisolvens and purified. The optimum pH for enzyme activity was 5.6. The enzyme preferentially degraded de-esterified substrates by hydrolysis of monosaccharide units from the non-reducing end; the only product of degradation was D-galacturonic acid. Values of Km and Vmax for oligo- and polygalacturonates indicated that the best substrate was digalacturonic acid; oligogalacturonates containing either a saturated or a delta 4,5-unsaturated non-reducing end were both degraded. The enzyme was classified as an exo-D-galacturonanase [poly(1,4-alpha-D-galacturonide) galacturonohydrolase (EC 3.2.1.67)].     

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.     

Kinetic attributes of Na+, K+ ATPase and lipid/phospholipid profiles of rat and human erythrocyte membrane

Kinetic properties of Na+, K+ ATPase of membranes from rat and human erythrocytes were examined. The enzyme stability decreased with incubation time. The Vmax of the human enzyme was about 4 times lower than the values of the rat enzyme. However the energies of activation were higher. Phase transition temperature for the rat and the human enzyme was 24 degrees C and 17 degrees C, respectively. The human erythrocyte membranes were characterized by lower total phospholipid and cholesterol contents and were relatively more fluid. The human membranes contained lower proportions of acidic phospholipids which correlated well with the lower Vmax of the enzyme; the proportion of lysophosphoglyceride and sphingomyelin was higher in the human membrane.     

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.     

Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease

Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.     

Influence of endothelial volume on kinetics of reacting indicators in the lung

The purpose of this work is to show mathematically the relationship between the classical maximum velocity of reaction, Vmax, for enzyme kinetics and an analogous parameter, Vmax, derived by Linehan and Dawson (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 47:404-411, 1979) for the analysis of tracers which disappear by saturation kinetics from the lung circulation during the passage of indicators after bolus injection. Rederivation of the original equation for the combination of flow and reaction in a capillary showed that Vmax is equal to the product of enzyme Vmax and the volume of endothelium, Ve, in which the enzyme resides. This implies that Vmax interpreted from multiple-indicator curves in the lung by the Linehan-Dawson method is a combination of an enzyme characteristic Vmax and a measure of functioning capillary surface during passage, Ve. Lung injury could change Vmax, functioning surface (Ve), or both.     

Regulation of UDP-glucuronosyltransferase by lipid-protein interactions. Comparison of the thermotropic properties of pure reconstituted enzyme with microsomal enzyme

The temperature dependence of two kinetic properties of the GT2P isoform of microsomal UDP-glucuronosyltransferase was studied for enzyme in intact microsomes and for pure enzyme reconstituted into different types of lipid bilayers. The properties studied were the non-Michaelis-Menten kinetics of the enzyme and activity at Vmax(app). For enzyme in intact microsomes, the pattern of non-Michaelis-Menten kinetics was seen at all temperatures in the range tested (23 to 48 degrees C), and the slopes of the Hill plots of the data were constant across this range of temperatures. Although non-Michaelis-Menten kinetics were present for pure enzyme in bilayers of 1,2-dimyristoylphosphatidylcholine or 1,2-dipalmitoylphosphatidylcholine only in the gel phase (Hockman, Y., Kelley, M., and Zakim, D. (1983) J. Biol. Chem. 258, 6509-6519), it was not possible to reconstitute this pattern of kinetics for enzyme at T greater than 40 degrees C. For example, GT2P displayed Michaelis-Menten kinetics in bilayers of 1,2-distearoylphosphatidylcholine at 44 degrees C. For enzyme in microsomes, activities at Vmax(app) increased with increasing temperature in the range 23 to 48 degrees C, with a discontinuity in the slope of the Arrhenius plot at 34 degrees C. This thermotropic property also could not be reconstituted with pure GT2P. Instead, activities at Vmax(app) for GT2P reconstituted in 1,2-dioleoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, or 1,2-stearoyl oleoylphosphatidylcholine increased in the range 23 to 37 degrees C, but then decreased at T greater than 37 degrees C. The fall in activity at T greater than 37 degrees C was reversible, indicating that GT2P undergoes a reversible change at 37 degrees C to a less active form of the enzyme. The differences between pure, reconstituted GT2P and microsomal GT2P indicate that the thermotropic properties of the microsomal enzyme are not properties of the enzyme per se but depend on interactions between it and other components in the microsome. The data suggest, therefore, that the properties of GT2P in microsomes results in part from an organization of components in the microsomal membrane.     

Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol-OT/isooctane microemulsions

Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol-OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH-NADH and by determination of Km,app and Vmax,app values for cyclohexanone. The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48-h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged. If no co-enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co-enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co-enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.     

Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.     

Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.     

Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors

To probe the active site of the heme protein indoleamine 2,3-dioxygenase, the effects of 3-indoleethanol (IET) (or tryptophol), one of the known indole derivative effectors, and indole (IND) on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption and CD) of the enzyme were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has shown that, at millimolar concentrations, both IET and IND lower considerably the Km value for D-Trp by approximately 25% and approximately 60%, respectively, at pH 7.0, while neither affects the Km value for L-Trp. Interestingly, however, these effectors exert opposite effects with respect to each other on the Vmax values for both D-Trp and L-Trp: IET enhances the Vmax values by 40-60% while IND lowers them by 12-24%. These effects of IET and IND on the Vmax values may be attributed to the shift in the ferric (inactive) enzyme----ferrous (active) enzyme equilibrium either to the right (IET) or to the left (IND) caused by the binding of these effectors to the enzyme in the steady state of the catalytic reaction. Both effectors induce clearly detectable spectral changes, especially notable in CD spectra, upon binding (in a 1:1 molar ratio, Kd = 10(-4) to 2.5 X 10(-3) M) to the ferrous enzyme and its complexes with O2, CO, and NO, both in the presence and in the absence of L-Trp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium and ATP affinities of the cardiac Na(+),K(+)-ATPase in spontaneously hypertensive rats

The Na(+),K(+)-ATPase is postulated to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembrane Na(+)-efflux from cardiac cells in spontaneously hypertensive rats (SHR). In the investigated group of SHR the systolic blood pressure and the heart weight were increased by 48% and by 60%, respectively. Upon activating the cardiac Na(+),K(+)-ATPase with substrate, its activity was lower in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (Vmax) by 28% which was accompanied with lowered affinity of the ATP-binding site as indicated by the increased value of Michaelis-Menten constant (Km) by 354% in SHR. During activation with Na(+), we observed an inhibition of the enzyme in hearts from SHR at all tested Na(+) concentrations. The value of Vmax decreased by 37%, and the concentration of Na(+) that gives half maximal reaction velocity (KNa) increased by 98%. This impairment in the affinity of the Na(+)-binding site together with decreased affinity to ATP in the molecule of the Na(+),K(+)-ATPase are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of SHR.     

Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47

Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis. The mutated enzymes were expressed in Escherichia coli and purified to homogeneity. The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively. The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1. The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase. In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains. The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes. Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme. The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

LmAPX基因在大肠杆菌和酵母菌中的表达研究

目的：研究枸杞抗坏血酸过氧化物酶基因（ascorbate peroxidase,LmAPX）在原核中的表达和酶学特性以及在酵母菌中的抗氧化能力,为进一步研究逆境诱导的抗氧化胁迫的作用机理奠定理论基础。方法：将LmAPX转入大肠杆菌BL21中进行异源表达,采用 Ni²⁺亲和层析,纯化重组蛋白,并对不同温度和pH值下的酶活进行研究,Lineweaver-Burk双倒数作图法测定该酶的Km和Vmax值。将LmAPX转入酵母菌株W303中进行H₂O₂和NaCl氧化胁迫处理。结果：该酶的最适温度和最适pH值分别为40℃和6.5。当抗坏血酸（Ascorbic acid,AsA）浓度过量时,对H₂O₂的Km和Vmax分别是0.17±0.02 mmol/L和11.78±1.88 mmol/min·mg;当H₂O₂浓度过量时,对AsA的Km和Vmax分别是2.19±0.40 mmol/L和58.82±3.51 mmol/min·mg。含有LmAPX基因的酵母菌株,在半乳糖的诱导下在8 mmol/L H₂O₂和100 mmol/L NaCl的培养基上的生长都明显优于对照组。结论：LmAPX蛋白具有很好的抗氧化性和耐盐性。     

Preparation and characterization of kappa-carrageenan immobilized urease

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.     

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.     

Inhibition of human natural killer activity by lysosomotropic agents

We have examined the effect of three lysosomotropic amines on human NK cell activity. Dansylcadaverine (DCA), diphenylamine (DPA), and lidocaine (LID) inhibited NK activity of nylon wool-purified and large granular lymphocyte (LGL)-enriched cell preparations. Cadaverine (CAD), an analog of DCA that does not affect lysosomal function, had no effect on NK activity. Binding of the K562 target cells to effector cells, as assessed in a single cell assay, was not inhibited by DCA, DPA, or LID. Cytotoxicity was inhibited by DCA and DPA only when these drugs were added within 5 min after the initiation of NK assays. In contrast, LID inhibited NK activity even when added 60 min after the addition of effector cells to target cells. All three amines that inhibited NK activity also reduced the intracellular concentration of the lysosomal enzyme beta-glucuronidase without affecting the activity of the cytoplasmic enzyme lactate dehydrogenase. Kinetic analysis revealed that LID inhibited both the maximum velocity (Vmax) of the cytotoxicity reaction as well as the affinity constant (Km); whereas DCA and DPA only inhibited Vmax.     

Calcineurin trapped in liposomes inhibits the action potentials of isolated 3-days-old embryonic chick ventricle.

Calcineurin, an intracellular protein phosphatase (type 2B), is reported to inhibit L-type (slow) calcium channels and thereby play a key role in channel inactivation. The present study was undertaken to examine effects of calcineurin on slow channel dependent action potentials of 3-days-old embryonic chick ventricle and to assess the role of this enzyme in regulation of developing slow channels. Calcineurin trapped in phosphatidylcholine-liposomes to facilitate its intracellular uptake was found to inhibit maximal upstroke velocity (+Vmax), overshoot and duration of action potentials. At higher doses of calcineurin containing liposomes the preparations ceased to exhibit spontaneous activity but elicited electrically driven action potentials with lower +Vmax and overshoot. These observations show that calcineurin down-modulates the embryonic cardiac slow channels under basal conditions.     

Isolation and characterization of an activator for Azotobacter vinelandii nicotinamide mononucleotide glycohydrolase

Azotobacter vinelandii NMN glycohydrolase [EC 3.2.2.14] has been shown to require absolutely GTP or a high-molecular-weight and heat-stable component for its function. The intracellular activator could be purified from its sonicate by heat treatment, acetone precipitation, phenol extraction, and acid precipitation in a good yield. The purified activator showed high affinity and effectiveness for NMN glycohydrolase (KA = 0.012 optical density unit at 257 nm/ml; Vmax standardized by the activity at 1 mM GTP = 88%). Negative cooperativity of the enzyme activation with the activator was also shown. On treatment with either micrococcal nuclease or pancreatic RNase, the activator activity was completely abolished, whereas pronase and trypsin had no effect. The activator could be replaced by yeast RNA as well as calf liver RNA, whereas DNAs purified from Micrococcus lysodeikticus, T 7 and calf thymus had no effect on the enzyme. Furthermore, poly(G) and poly(I) could function as activators with the same effectiveness as the purified activator, and the enzyme activation with these RNA homopolymers was inhibited by poly(C), suggesting that the activation mechanism is specific with respect to base composition. Based on a kinetic analysis of the enzyme activation with commercial RNAs, together with the results from enzymatic digestion, specific inhibition of the enzyme by spermine, and its chemical properties, the activator was identified as an RNA. A model is described for NMN glycohydrolase regulation in which the RNA activator plays an important role in the NMN salvage cycles.