5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs

The substrate specificity of isoleucyl-tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl-tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres. The order of substrate addition to isoleucyl-tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl-tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E . Ile-AMP . ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid-equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady-state ordered ter-ter mechanism; in the presence of pyrophosphatase the mechanism is bi-uni uni-bi ping-pong similarly as observed for the yeast enzyme. A comparison of inhibition patterns obtained with N(6)-benzyladenosine 5'-triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist. The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E . tRNA . Ile-AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping-pong mechanism.     

Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity

The nucleotide-sugar pyrophosphatase activity of rat small intestine was studied using GDP-[14C]Man as substrate. The highest specific activities in the gastrointestinal tract were in the proximal small intestine, with a preferential localization in villus tip cells. Purified brush-border membranes were highly enriched in nucleotide-sugar pyrophosphatase. After the enzyme was solubilized with detergent and purified 180-fold, it hydrolyzed FAD and p-nitrophenyl-5'-thymidylate, as well as nucleotide sugars. That the same enzyme, a 5'-nucleotide phosphodiesterase, is responsible for nucleotide-sugar pyrophosphatase, phosphodiesterase I, and FAD pyrophosphatase activities is indicated by: co-migration in electrophoresis, parallel thermal inactivation, competitive inhibition studies, and similar regional, cellular, and subcellular localizations.     

Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.

1,N6-Etheno derivatives of pyridine analogues of NAD+ were synthesized, characterized and tested as substrates for a fluorimetric assay of nucleotide pyrophosphatase (EC 3.6.1.9). Upon cleavage of their pyrophosphate bond, the fluorescence of pyridine-1,N6-ethenoadenine dinucleotide (epsilon PdAD+) and of 4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (epsilon hy4PdAD+) increased respectively 15-and 73-fold, at pH 7.4. This property allows a convenient steady-state assay of nucleotide pyrophosphatase by continuous monitoring of reaction progress. Both compounds were good substrates of this class of enzyme. The relative insensitivity of the fluorescence of epsilon PdAD+ and epsilon hy4PdAD+ to pH changes allowed assays under conditions preserving cellular integrity. epsilon PdAD+ is useful as a substrate for measuring nucleotide pyrophosphatase activity on the outside of mammalian cells because it is not a substrate for the external NAD+ glycohydrolase. epsilon Hy4PdAD+ proved useful when high sensitivity was needed.     

Inhibition studies on Rhodospirillum rubrum H(+)-pyrophosphatase expressed in Escherichia coli

The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N,N'-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5'-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg(2)PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg(2+), possibly reflecting a change in substrate preference from Mg(2)PPi to MgPPi. BPB is a H(+)-pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support

A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli. Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol. In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding. The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase. Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity.     

Inorganic pyrophosphatase as a label in heterogeneous enzyme immunoassay

Inorganic pyrophosphatase from Escherichia coli has been employed as a label in heterogeneous enzyme immunoassays. Enzyme-antibody conjugates were prepared with the use of glutaraldehyde and purified by gel permeation chromatography. Enzyme activity was measured by means of a sensitive one-step color reaction between phosphate, molybdate, and malachite green. The sensitivity in terms of absorbance readings was four to eight times higher than that of peroxidase-based assays. The color change (yellow to greenish blue) inherent in the use of pyrophosphatase as the labeling agent is highly suitable for visual analysis. Other merits of pyrophosphatase include the remarkable stability of the enzyme and its substrate, its compatibility with bacteriostatic agents, and its low Michaelis constant. Examples of the use of phosphatase in the assay of human alpha-fetoprotein and immunoglobulin G are presented.     

Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast]

A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done. It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator. Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate. The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase. The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.     

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

Galactosyltransferase, pyrophosphatase and phosphatase activities in luminal plasma of the cauda epididymidis and in the rete testis fluid of some mammals

Galactosyltransferase activity was measured in the luminal plasma of the cauda epididymidis of mice, rats, rabbits, rams and boars, and in the rete testis fluid of rams and boars. The activities of nucleotide pyrophosphatase and alkaline phosphatase, which compete with galactosyltransferase for substrate, were also determined. In these species, galactosyltransferase activity in the luminal plasma of the cauda epididymidis was similar when the inhibitory effect of pyrophosphatase and phosphatase was minimized by assay conditions. However, under assay conditions that did not minimize the effect of these enzymes, the galactosyltransferase activities of these species were very different and were inversely correlated with the activities of pyrophosphatase and phosphatase. The ratio of galactosyltransferase activity to pyrophosphatase and phosphatase activity was much higher in the rete testis fluid than in the luminal plasma of the cauda epididymidis in both rams and boars. In rams, galactosyltransferase in the luminal plasma of the cauda epididymidis was more heat resistant than that in serum. These results suggest that there is a species difference in the availability of galactosyltransferase activity in the luminal plasma of the cauda epididymidis and that in some species, galactosyltransferase in the luminal fluid is unlikely to have any function. The results are also discussed with respect to the possible function of galactosyltransferase, pyrophosphatase and phosphatase in epididymal luminal plasma and rete testis fluid.     

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.     

Mutational analysis of substrate recognition by human arginase type I--agmatinase activity of the N130D variant

Upon mutation of Asn130 to aspartate, the catalytic activity of human arginase I was reduced to approximately 17% of wild-type activity, the Km value for arginine was increased approximately 9-fold, and the kcat/Km value was reduced approximately 50-fold. The kinetic properties were much less affected by replacement of Asn130 with glutamine. In contrast with the wild-type and N130Q enzymes, the N130D variant was active not only on arginine but also on its decarboxylated derivative, agmatine. Moreover, it exhibited no preferential substrate specificity for arginine over agmatine (kcat/Km values of 2.48 x 10(3) M(-1) x s(-1) and 2.14 x 10(3) M(-1) x s(-1), respectively). After dialysis against EDTA and assay in the absence of added Mn2+, the N130D mutant enzyme was inactive, whereas about 50% full activity was expressed by the wild-type and N130Q variants. Mutations were not accompanied by changes in the tryptophan fluorescence properties, thermal stability or chromatographic behavior of the enzyme. An active site conformational change is proposed as an explanation for the altered substrate specificity and low catalytic efficiency of the N130D variant.     

Identification of an extracellular nucleotide pyrophosphatase in the culture media of Streptomyces mediterranei ME/R 17]

An exocellular pyrophosphatase, active on the nucleotide precursors of peptidoglycans, has been found in the culture medium of Streptomyces mediterranei ME/R 17. This enzyme was separated from the DD-carboxypeptidase by batchwise adsorption on DEAE cellulose. The pyrophosphatase had no strict substrate requirements, it hydrolyzed various UDP-sugar substrates: UDP-GlcNAc, UDP-Mur NAc and UDP-MurNAc peptides, giving rise to the corresponding sugar phosphate and to UMP. The enzyme preparation also contained a 5'-nucleotidase activity and UMP was further split to give uridine. This nucleotidase activity was inhibited by potassium tetraborate. Both cytoplasmic and particulate preparations from cells of S. mediterranei also contained a pyrophosphatase activity while only the particulate fractions showed the DD-carboxypeptidase activity. The pyrophosphatase excretion was tested during the grwoth cycle. The activity of the enzyme showed a constant increase throughout the exponential growth and a stronger increase in the late exponential phase. Such a result could be correlated with a consumption of the nutrients in the culture medium, in fact a relatively poor culture medium had a strong positive effect upon the production of the exocellular pyrophosphatase.     

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.     

Analysis of a conserved arginine R281L in catalysis in Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase is essential for the catalytic transformation of a linear tripeptide substrate δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N in the biosynthesis of β-lactam antibiotics. The recent Aspergillus nidulans isopenicillin N synthase crystal structure proposed that a conserved arginine, R279, has a role in substrate binding. This study, the first site-directed mutagenesis experiment on arginine in isopenicillin N synthase, was carried out to ascertain the role of the similarly conserved and corresponding arginine residue R281 on catalysis in the fungal Cephalosporium acremonium isopenicillin N synthase. Replacement of the arginine residue with leucine to generate the mutant R281L Cephalosporium isopenicillin N synthase resulted in undetectable activity as shown by enzyme bioassays. It is possible that the mutant's substrate binding capability was eliminated, thus preventing the catalytic reaction. Further investigation into the corresponding arginine residues in isopenicillin N synthase of other species is warranted.     

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.