Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with $\text{bis-(}\text{p-}\text{nitrophenyl)}$ phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards $\text{5′ -}\text{dTMP}\text{-p-}\text{nitrophenyl}$ ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography.

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.     

Characterization of phosphohydrolase activity in bovine spleen extracts: identification of a bis(p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate

Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).     

Enzymatic and chemical analyses of pig platelet membrane subfractions isolated by zonal centrifugation.

1. A mixed membrane fraction prepared from pig platelets was subfractionated, using the "B 14" zonal rotor, into two distinct subpopulations of membrane vesicles, each associated with a different phosphodiesterase activity. 2. The lighter subfraction (MI) was enriched 7-8 fold with bis-(p-nitrophenyl) phosphate phosphodiesterase activity and the denser subfraction (MII) showed a similar degree of enrichment of 5'dTMP-p-nitrophenyl ester phosphodiesterase activity. 3. Assays for other enzyme activities revealed slight enrichement (approx. 2 fold) of acid phosphatase, 3'-dTMP-p-nitrophenyl ester phosphodiesterase and beta-glucuronidase activities in MI, and beta-galactosidase in MII. Cyclic AMP phosphodiesterase, lactate dehydrogenase and N-acetyl-beta-glucosaminidase showed negligible activity in both MI and MII, and succinate dehydrogenase activity could not be detected in either subfraction. 4. Chemical analyses of the membrane subfractions demonstrated that MI contained approx. twice as much cholesterol, phospholipid, sialic acid and hexosamine per unit weight of protein than MII. These results are consistent with our previously reported observations from surface-labelling experiments, which indicated that MI was derived principally from the platelet surface-exposed membranes and that MII was probably intracellular in origin. 5. Analysis of the membrane polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the presence of 12-15 components, in each subfraction, in the mol. wt. range 12000-200000, including a prominent band of approx. mol. wt. 46000, which has beeen identified to be actin. Qualitative as well as possible quantitative differences were apparent in that MII contained three components in addition to those present in MI. 6. Analysis of the periodate-Schiff staining components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated the presence of 4 major glycoproteins in both subfractions with apparent mol. wt. ranging from approx. 95000 to 150000; in addition two minor components were also present. Further, a very fast-migrating band, which did not stain with Coomassie blue, was observed in both MI and MII and probably represents lipid material.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.     

Sphingomyelinase and nonspecific phosphodiesterase activities in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease A, B and C

Acid sphingomyelinase activity determined using the natural substrate, [choline-methyl-14C]sphingomyelin, or the chromogenic synthetic analogue, 2-N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine, was deficient in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease types A and B. In contrast, lines from Niemann-Pick disease type C and "sea-blue histiocyte syndrome" showed a sphingomyelinase activity within the normal range. Bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)pyrophosphate phosphodiesterase activities were not deficient in any Niemann-Pick disease cell line. These results demonstrate the validity of such cell lines as an experimental model system for enzymatic studies of Niemann-Pick disease.     

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity.

This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described.     

Extracellular enzyme activities associated with epiphytic microbiota on submerged stems of the reed Phragmites australis

Abstract Fluorogenic 4-methylumbelliferyl (MUF) compounds were used as analogue substrates for assay of extracellular enzyme activities associated with epiphytic microbiota at submerged Phragmites australis stem surfaces. Incubations at a range of MUF substrate concentrations indicated that saturation of enzyme activity was achieved at a MUF substrate concentration of about 200 μmol 1⁻¹. Later determinations at a single, saturation, concentration of MUF substrate were, therefore, carried out at about 200 μmol l⁻¹. Such determinations were undertaken using P. australis stems from eight gravel-pit ponds. The rate of enzymatic hydrolysis of MUF phosphate (analogue substrate for phosphatase activity) was > MUF β- d -glucopyranoside (β- d -glucosidase) > MUF β- d -galactopyranoside (β- d -galactosidase) > MUF sulphate (sulphatase) and MUF palmitate (lipase) on stems from all eight ponds. Thus the relative magnitude of the various components of total epiphyton extracellular enzyme activity might be a conservative feature.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Isolation of human platelet plasma membranes with polylysine beads

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.     

Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester

The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Studies on alkaline phosphatase: Inhibition by phosphate derivatives and the substrate specificity

1. The kinetics of inhibition of calf-intestinal alkaline phosphatase by inorganic phosphate, fluorophosphate, inorganic pyrophosphate, beta-glycerophosphate and adenosine 5'-triphosphate in the range pH8-10 were investigated. The reference substrate was 4-methylumbelliferyl phosphate. 2. The inhibitions were ;mixed' in that both K(m) and V were affected, but the competitive element was by far the stronger. 3. In each case the pH profile for the competitive K(i) was similar to the pH profile for K(m). Since the K(m) and K(i) values both change 100-fold over the pH range 8-10, it is concluded that the inhibitors compete with the substrate for the same active site. 4. It was also found that the enzyme preparation hydrolysed fluorophosphate, pyrophosphate and adenosine 5'-triphosphate as readily as it hydrolysed 4-methylumbelliferyl phosphate and beta-glycerophosphate. Each pH-activity curve, however, had a different shape, but with the exception of pyrophosphate the activity approached the same maximum value at high pH. 5. Attempts to separate the phosphomonoesterase and pyrophosphatase activities by column chromatography were not successful, and the results of other experiments listed suggest that the two activities are a property of the same enzyme. 6. The effect of Mg(2+) ions is briefly mentioned: the phosphomonoesterase activity is enhanced whereas the pyrophosphatase and adenosine triphosphatase activities are strongly inhibited in the presence of excess of Mg(2+) ions.     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

Determination of phosphodiesterase activity in the presence of phosphomonoesterase using bis-p-Nitrophenyl phosphate

In the presence of phosphomonoesterase contaminations the use of bis-p-nitrophenyl phosphate to measure phosphodiesterase activity gives incoclusive values because one of the products of the phosphodiesterase or nuclease reaction becomes a substrate of the contaminating enzyme. A direct determination of the hydrolyzed phosphodiesterase substrate in the UV range is possible at the isosbestic points of the transformation of the phosphomonoesterase substrate.     

The properties of alpha-galactosidase remaining in kidney and liver of patients with Fabry's disease

α-Galactosidase activity is diminished in the kidney and liver of patients with Fabry's disease. Less than 2% of the normal activity was found in their kidney, while more than 20% of the normal activity was retained in their liver. The residual enzyme in these two organs showed a single component of pI 4.5 with activity toward 4-methylumbelliferyl α-galactoside on isoelectric focusing. This component seemed to correspond to Fr. II of normal liver or kidney. Ceramide trihexosidase activity was observed as a single component in the same fractions as the α-galactosidase activity for the synthetic substrate.In normal liver, 4-methylumbelliferyl α-galactoside hydrolase was separated into four components with pI's of 4.9, 4.5, 4.2 and 3.9 by isoelectric focusing. Fr. II with pI 4.5 differed from Fr. I in its heat stability and inhibition by myoinositol. In spite of some dissimilarities in their properties, the ratios of enzyme activities for ceramide trihexoside and 4-methylumbelliferyl α-galactoside were similar in all the components of both normal liver and kidney.     

Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon

The promoter-proximal gene (glpT) of the glpT-glpQ operon of Escherichia coli encodes a membrane permease responsible for active transport of sn-glycerol 3-phosphate. Promoter-distal glpQ encodes a periplasmic protein which is not required for active transport of sn-glycerol 3-phosphate (Larson, T.J., Schumacher, G., and Boos, W. (1982) J. Bacteriol. 152, 1008-1021). This periplasmic protein has now been identified as a phosphodiesterase which hydrolyzes glycerophosphodiesters into sn-glycerol 3-phosphate plus alcohol. The enzyme exhibited broad substrate specificity with respect to the alcohol moiety; sn-glycerol 3-phosphate was released from glycerophosphoethanolamine, glycerophosphocholine, glycerophosphoglycerol, and bis(glycerophospho)glycerol. The enzyme was specific for glycerophosphodiesters; bis(p-nitrophenyl)phosphate, a substrate for other phosphodiesterases, was not hydrolyzed. In a coupled spectrophotometric assay utilizing sn-glycerol 3-phosphate dehydrogenase and NAD, apparent activity was optimal at pH 9 and was stimulated by Ca2+. The substrates of the phosphodiesterase had no affinity for the glpT-encoded active transport system. Thus, the glpQ gene product expands the catabolic capability of the glp regulon to include a variety of glycerophosphodiesters.