Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization.

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning,Expression in Escherichia coli,and Biochemical Characterization of the Recombinant Protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60–65°C, pH 11.0, K _M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg²⁺ and declined in the presence of EDTA.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

K+-and Mg2+-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca²⁺ + Mg²⁺-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg²⁺ and EGTA and is stimulated by Ca²⁺. The Mg²⁺-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl₂, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca²⁺ causes no further increase in the rate of hydrolysis and Ca²⁺ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca²⁺ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by P_i unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect P_i inhibition of the Mg²⁺-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a P_i-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and P_i. In this conformation the enzyme is transformed from a Ca²⁺- and Mg²⁺-dependent ATPase into a (K⁺ + Mg²⁺)-ATPase.     

Regulation in the chemolithotrophthiobacillus neapolitanus: Fructose-1,6-diphosphatase

Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10^–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10^–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect.     

SSF production,purification and characterization of chitin deacetylase from Aspergillus flavus

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn²⁺ and Zn²⁺. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The K_m and k_cat values of the enzyme were determined to be 9.45?mg mL^?1 and 26.72?s^?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Purification and properties of isocitrate lyase from Candida brassicae E-17

Isocitrate lyase (EC 4.1.3.1) was purified from acetate-grown cells of Candida brassicae E-17, by ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 gel filtration column chromatographies. The purified enzyme was electrophoretically homogeneous. The molecular weight of this enzyme was 290,000 by gel filtration, and it was composed of four identical subunits whose molecular weights were 71,000 each. The pH and temperature optima were 6.8 and 37°C, respectively. The enzyme was stable from pH 6.0 to 7.0. The enzyme was activated by Mg²⁺ and the maximum activity was obtained with a concentration of 8 mM Mg²⁺. The enzyme was also activated by Mn²⁺ and Ba²⁺. The activity of this enzyme was stimulated by reducing agents. The K_m values for dl-isocitrate were 1.5 mM in sodium phosphate buffer and 0.62 mM in imidazole-HCl buffer.     

Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes

Summary Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES¹ buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES² or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration.In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.1 PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - 2 HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid This study was supported by a Grant-in Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture, the Japanese Government (No. 57770012)     

Properties and partial purification of mevalonate kinase from Agave americana

Mevalonate kinase activity was demonstrated in acetone powder extracts from Agave americana leaves, flowers and scape. ATP was the most effective phosphate donor. The enzyme had an optimum pH of 7.9 in Tris-HCl buffer. Dialysis decreased the ability to phosphorylate mevalonic acid (MVA). Partially purified mevalonate kinase reached maximum activity in the presence of 2 mM Mn²⁺ or 6–8 mM Mg²⁺. Higher concentrations of Mn²⁺ were inhibitory, whereas higher concentrations of Mg²⁺ produced only a small decrease in the activity. The amount of mevalonate-5-phosphate (MVAP) formed depended on protein concentration and incubation time. During short incubations, the MVAP formed increased as protein concentration rose, whereas during prolonged incubations (1–6 hr), there was a decrease in the MVAP formed when a certain amount of protein was exceeded. It is suggested that MVAP formed was hydrolysed by a phosphatase present in the extracts. This interfering activity was eliminated when mevalonate kinase is partially purified. The apparent K_m values of the enzyme from leaves were 0.05 mM for MVA and 0. 14 mM for ATP. Similar K_m values are obtained with partially purified mevalonate kinase. The enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 filtration and DEAE-Sephadex A-50 fractionation.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.