Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella tertiolecta cells

Glycerol-3-phosphatase (EC 3.1.3.2.1) was studied by following the release of radioactive glycerol from L-(U-¹⁴C)glycerol-3-phosphate in Dunaliella tertiolecta enzyme extracts. The reaction showed a neutral pH optimum and had an absolute requirement for Mg²⁺. The substrate saturation curve was hyperbolic with an apparent K _m value for glycerol-3-phosphate of 0.7 mM in the absence of phosphate. Inorganic orthophosphate was a competitive inhibitor of the enzyme with an estimated K _j of 0.1 mM. The glycerol-3-phosphatase reaction was blocked nearly completely by millimolar Ca²⁺ concentrations. Ca²⁺ inhibition did not depend on the presence of calmodulin in the reaction medium. The characteristics of glycerol-3-phosphatase are discussed in relation to the regulation of the cyclic glycerol metabolism in Dunaliella cells during periods of osmotic stress.     

Calcium-ion transport by intact Ehrlich ascites tumor cells at 0°C

At 0°C, where Ca²⁺ efflux is not observed, the uptake of Ca²⁺ by Ehrlich ascites tumor cells consists of four components: 1) An energy-dependent mitochondrial component, which is inhibited by uncouplers, respiratory inhibitors, and mitochondrial ATP-ase inhibitors. 2) Binding to the cell surface, which can be displaced by an EGTA wash. 3) An electrochemical gradient-dependent component, which is inhibited by agents which dissipate these gradients, such as proton ionophores, metabolic uncouplers, and valinomycin. The valinomycin inhibition of this transport component is dependent on K⁺ concentration. 4) Passive diffusion, which is dependent on Ca²⁺ concentration and is observed in the presence of inhibitors of the other components. The uptake of Ca²⁺ at 0°C is sensitive to ruthenium red presumably due to its competition with Ca²⁺ for cell binding sites.     

Properties of a purified NAD-linked alpha-glycerophosphate dehydrogenase from Leptomonas sp.; activation by polyamines.

The respiration of rat heart mitochondria incubated with EGTA fails to respond to the addition of uncouplers when β-hydroxybutyric acid is the substrate. By contrast, the addition of ADP and phosphate is followed by the normal State 4/State 3 transition. The phenomenon is due to the complete loss of Ca²⁺ from mitochondria induced by uncouplers and EGTA, and can indeed be duplicated by incubation in the presence of the specific Ca²⁺ ionophore A23187 and ruthenium red (the latter prevents the re-uptake of the lost Ca²⁺). Since the loss of Ca²⁺ has no effect on the oxidation of other NAD-dependent substrates, it is concluded that Ca²⁺ is essential for the interaction of β-hydroxybutyric acid dehydrogenase with a specific intramembrane $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ pool.     

Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus)

We isolated calcium-labile mitotic spindles from eggs of the sea urchin Lytechinus variegatus, using a low ionic strength, EGTA lysis buffer that contined 5.0 mM EGTA, 0.5 mM MgCl2, 10-50 mM PIPES, pH 6.8, with 1% Nonidet P-40 (detergent) and 20-25% glycerol. Isolated spindles were stored in EGTA buffer with 50% glycerol for 5-6 wk without deterioration. The isolated spindles were composed primarily of microtubules with the chromosomes attached. No membranes were seen. Isolated spindles, perfused with EGTA buffer to remove the detergent and glycerol, had essentially the same birefringent retardation (BR) as spindles in vivo at the same mitotic stage. Even in the absence of glycerol and exogenous tubulin, the isolated spindles were relatively stable in the EGTA buffer: BR decayed slowly to about half the initial value within 30-45 min. However, both the rate and extent of BR decay increased with concentrations of Ca2+ above 0.2-0.5 muM as assayed using Ca-EGTA buffers (0.2 mM EGTA, 0.5 mM MgCl2, 50 mM PIPES, pH 6.8, plus various amounts of CaCl2). Microtubules depolymerized almost completely in < 6 min at Ca2+ concentrations of 2 muM and within several seconds at 10 muM Ca2+. Of several divalent cations tested, only Sr2+ caused comparable changes in BR. The absence of membranes in the isolated spindles appeared to be associated with a lack of calcium- sequestering ability. Our results suggest that calcium ions play an important role in the depolymerization of spindle microtubules and that membrane components may function within the mitotic apparatus of living cells to sequester and release calcium ions during mitosis.     

Glycerol-3-phosphatase of Corynebacterium glutamicum

Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg2? or Mn2? for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg?1 with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and shown to significantly increase GPP activity.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.     

Further observations on calcium and other divalent cations metabolism in intact Ehrlich ascites tumour cells

Summary The metabolism of calcium has been investigated in the Ehrlich Ascites Tumour Cells (ATC). ATC extrude Ca²⁺ actively by an energy-dependent mechanism, supported by both respiration and glycolysis. Extrusion takes place even against a very steep concentration gradient (10 mM Ca²⁺). Cell calcium content is decreased by monovalent cations (Na⁺, K⁺ and Li⁺), which act independently from their metabolic effects. La³⁺ inhibits ATC Ca²⁺ extrusion whereas Ruthenium Red slightly decreases cell calcium content. The antibiotic ionophore A 23187 strongly increases ATC Ca²⁺ level. The metabolism of other divalent cations (Mg²⁺, Sr²⁺ and Mn²⁺) has been studied. Mg²⁺ does not show appreciable changes in the various metabolic conditions tested, while Mn²⁺ and Sr²⁺ behave quite differently from Ca²⁺, suggesting a different distribution of these cations in ATC. The experimental findings indicate that Ehrlich Ascites Tumour Cells regulate their calcium content by mechanisms related to plasma membranes while the size and activity of mitochondrial compartment is of minor importance.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

Uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of Neurospora crassa

Summary Seven mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. One strain was deficient for NAD-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. Glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mM glycerol, but at higher concentrations free diffusion predominated. Glycerol uptake was decreased by cycloheximide and was more sensitive to sodium azide than to iodoacetate.     

Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (mucolipidosis II).

The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg⁺⁺ and ruthenium red. Diamide potentiates the swelling action of Ca⁺⁺, while DTE potentiates that of Mg⁺⁺. The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg⁺⁺. The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca⁺⁺ and Mg⁺⁺ is closely correlated with the redox state of membrane bound thiol groups.     

Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

Spatial structures in mitochondrial suspension induced by cation efflux

The formation of spatial structures in a thin unstirred layer of a mitochondrial suspension has been studied. It is shown that the structure formation depends on the state of the ion-transporting systems of mitochondria and that pattern development coincides with the activation of cation efflux from preloaded mitochondria. Spatial structure formation is an energy-dependent process and is suppressed by respiratory chain inhibitors. Patterning is also inhibited by EGTA, EDTA and ruthenium red, reflecting the requirement for divalent cation translocation in mitochondria for the studied phenomenon.     

Characterization of the Glycerolipid Composition and Biosynthetic Capacity of Pea Root Plastids

The glycerolipid composition of pea (Pisum sativum L.) root plastids and their capacity to synthesize glycerolipids from [UL-14C]glycerol-3-phosphate were determined. Pea root plastids primarily consist of monogalactosyldiacylglycerol, triacylglycerol, phosphatidylcholine, digalactosyldiacylglycerol, and diacylglycerol. Maximum rates of total glycerolipid biosynthesis were obtained in the presence of 2.4 mM glycerol-3-phosphate, 15 mM KHCO3, 0.2 mM sodium-acetate, 0.5 mM each of NADH and NADPH, 0.05 mM coenzyme A, 2 mM MgCl2, 1 mM ATP, 0.1 M Bis-Tris propane (pH 7.5), and 0.31 M sorbitol. Glycerolipid biosynthesis was completely dependent on exogenously supplied ATP, coenzyme A, and a divalent cation, whereas the remaining cofactors improved their activity from 1.3- to 2.4-fold. Radioactivity from glycerol-3-phosphate was recovered predominantly in phosphatidic acid, phosphatidylglycerol, diacylglycerol, and triacylglycerol with lesser amounts in phosphatidylcholine and monoacylglycerol. The proportions of the various radiolabeled lipids that accumulated were dependent on the pH and the concentration of ATP and glycerol-3-phosphate. The data presented indicate that pea root plastids can synthesize almost all of their component glycerolipids and that glycerolipid biosynthesis is tightly coupled to de novo fatty acid biosynthesis. pH and the availability of ATP may have important roles in the regulation of lipid biosynthesis at the levels of phosphatidic acid phosphatase and in the reactions that are involved in phosphatidylglycerol and triacylglycerol biosynthesis.     

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.     

D-Glucosaminate Aldolase Activity of D-Glucosaminate Dehydratase from Pseudomonas fluorescens and Its Requirement for Mn2+ Ion

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-o-gluconate and ammonia: α,β-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3–4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn²⁺ + pyridoxal 5′-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn²⁺ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn²⁺, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn²⁺ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress

Abstract Dunaliella tertiolecta cells subjected to a hyperosmotic shock of 0.930 osmol kg^?1 start almost immediately to synthesize glycerol at a rate of some 100 nmol min^?1 mg protein^?1. Glycerol synthesis was equally fast in both light and darkness, and was not affected by the nature but only by the concentration of solutes. During the period of rapid glycerol synthesis, which lasted about 1h, the concentration of glycerol-3-phosphate transiently increased. During the same period, ATP, fructose 1-6-bisphosphate, and triosephosphate content decreased markedly, especially when 0.1 kmol m^?3 NaCl-grown cells were used. The content of hexose-6-phosphates, nicotinamide coenzymes, and phosphate underwent no dramatic changes. Since no in vitro activity changes of the glycerol cycle enzymes could be detected during the adaptation period, the activity of glycerol-3-phosphate dehydrogenase in vivo is probably increased by a change in concentration of its effectors such as ATP.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight