Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

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.     

The localization of glycerol-3-phosphate dehydrogenase inEscherichia coli

Summary Starved cells ofEscherichia coli are dependent on an exogenous source of energy. It was of interest to ask whether compounds that are commonly used to supply energy must themselves be transported or whether they can be utilized on the outer portion of the cytoplasmic membrane. The utilization of glycerol-3-phosphate an energy source is totally dependent on the membrane-bound glycerol-3-phosphate dehydrogenase. In the present report glycerol-3-phosphate was used as the energy source for uptake of amino acids. A mutant was constructed which is unable to transport this ester and the starved mutant could not drive the uptake of glutamine with glycerol-3-phosphate. It is concluded that the enzyme is located on the internal surface of the membrane in intactE. coli cells. Further evidence was obtained by showing that no glycerol-3-phosphate dehydrogenase activity could be measured in either intact cells or spheroplasts using ferricyanide as electron acceptor, due to its impermeability. The activity could be measured after destruction of the membrane permeability barrier by toluenization. With membrane vesicles prepared according to Kaback's procedure nearly half of the dehydrogenase activity was accessible to ferricyanide as well as to impermeable competitive inhibitors of the enzyme. Partial inversion during preparation of vesicles is the most probable explanation for the results.A protion of this work was presented at the Miami Winter Symposia on the Molecular Basis of Biological Transprot, 1972.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Substrate-induced dissociation of glycerol-3-phosphate dehydrogenase and its complex formation with fructose-bisphosphate aldolase

A threefold decrease in specific activity of glycerol-3-phosphate dehydrogenase was found on going from 800 nM to 10 nM enzyme concentration. According to ultracentrifugal analyses the dimeric glycerol-3-phosphate dehydrogenase (molecular weight 78,000) dissociates into monomers in the equilibrium mixture of its substrates and products. The concentration-dependent decrease in the specific activity is interpreted as a consequence of subunit dissociation and the estimated dissociation constants are 0.7 micro M and 3.5 micro M at 38 degrees C and 20 degrees C respectively. According to active-enzyme-band centrifugation experiments and kinetic analysis aldolase forms a complex with glycerol-3-phosphate dehydrogenase and this complex formation influences the specific activity of the dehydrogenase. The interaction between glycerol-3-phosphate dehydrogenase and aldolase can provide a regulatory mechanism at the branching point of glycolytic and lipid metabolic pathways.     

The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat]

The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.     

Relationship between glycerol-3-phosphate dehydrogenase,fatty acid synthase and fatty acid binding proteins in developing human placenta

The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.     

Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter

Previous attempts to manipulate oil synthesis in plants have mainly concentrated on the genes involved in the biosynthesis and use of fatty acids, neglecting the possible role of glycerol-3-phosphate supply on the rate of triacylglycerol synthesis. In this study, a yeast gene coding for cytosolic glycerol-3-phosphate dehydrogenase ( gpd 1) was expressed in transgenic oil-seed rape under the control of the seed-specific napin promoter. It was found that a twofold increase in glycerol-3-phosphate dehydrogenase activity led to a three- to fourfold increase in the level of glycerol-3-phosphate in developing seeds, resulting in a 40% increase in the final lipid content of the seed, with the protein content remaining substantially unchanged. This was accompanied by a decrease in the glycolytic intermediate dihydroxyacetone phosphate, the direct precursor of glycerol-3-phosphate dehydrogenase. The levels of sucrose and various metabolites in the pathway from sucrose to fatty acids remained unaltered. The results show that glycerol-3-phosphate supply co-limits oil accumulation in developing seeds. This has important implications for strategies that aim to increase the overall level of oil in commercial oil-seed crops for use as a renewable alternative to petrol.     

Enzyme activity and content of glycerophosphate shuttle metabolites in the cortex and medulla oblongata during hyperbaric oxygenation therapy of acute hemorrhage

The experiments on cats have demonstrated that hyperbaric oxygenation (303.9 kPa, 60 min) in acute blood loss (24.0 +/- 0.8 ml/kg) has an essential effect on glycerophosphate shuttle mechanism: it stimulates mitochondrial glycerol-3-phosphate dehydrogenase in the sensorimotor cortex and medulla oblongata, providing glycerol-3-phosphate dehydrogenation, activates cytoplasm hydrogen delivery to mitochondrial respiratory chain, prevents glycerol-3-phosphate and NADH level increase and cytoplasm glycerol-3-phosphate dehydrogenase inhibition, limiting lactate production.     

盐生杜氏藻3-磷酸甘油脱氢酶基因克隆及其蛋白质结构预测

本文通过对盐生杜氏藻(Dunaliella salina) cDNA文库进行大规模EST测序并结合RACE实验克隆了盐生杜氏藻3-磷酸甘油脱氢酶基因, 并且通过Southern blotting实验确定了该基因在这个物种中拷贝数。通过生物信息学的方法, 预测该基因所编码的蛋白质结构, 验证了该蛋白质具有3-磷酸甘油脱氢酶完整的功能性结构域和磷酸水解酶类结构域。本实验为解释盐生杜氏藻在面临高渗胁迫下快速合成甘油的机制提供了帮助。     

盐生杜氏藻3-磷酸甘油脱氢酶基因克隆及其蛋白质结构预测

本文通过对盐生杜氏藻（Dunaliella salina）cDNA文库进行大规模EST测序并结合RACE实验克隆了盐生杜氏藻3-磷酸甘油脱氢酶基因,并且通过Southern blotting实验确定了该基因在这个物种中拷贝数。通过生物信息学的方法,预测该基因所编码的蛋白质结构,验证了该蛋白质具有3-磷酸甘油脱氢酶完整的功能性结构域和磷酸水解酶类结构域。本实验为解释盐生杜氏藻在面临高渗胁迫下快速合成甘油的机制提供了帮助。     

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1)

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.     

Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno-and enzyme histochemical study

Summary The differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in bovine hearts was studied with specific antibodies against M-line proteins (MM-creatine kinase and myomesin) and with enzyme histochemistry (succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase). MM-creatine kinase was detected at an earlier stage in Purkinje fibres and atrial myocytes than in ordinary ventricular myocytes. The findings are in agreement with previous ultrastructural observations that an earlier appearance of a dense M-band occurs in Purkinje fibres than in ordinary ventricular myocytes. Myomesin was detected in all three cell types even at early foetal stages, in accordance with suggestions that it is an integral component of the myofibrillar structure. The activity of succinate dehydrogenase gradually increased in both ordinary ventricular and atrial myocytes, while the activity of mitochondrial glycerol-3-phosphate dehydrogenase was high at different stages of early foetal development in the two tissues, finally becoming low in the adult stage. The activity of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase seemed to remain unchanged in the Purkinje fibres from early to late foetal stages. The present study shows that the Purkinje fibres are already different from ordinary ventricular myocytes at early foetal stages and that the two cell types differentiate in different ways. It is concluded that there are also developmental differences between ordinary ventricular and atrial myocytes.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

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     

Nucleotide sequence and gene-polypeptide relationships of the glpABC operon encoding the anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12.

The nucleotide sequence of a 4.8-kilobase SacII-PstI fragment encoding the anaerobic glycerol-3-phosphate dehydrogenase operon of Escherichia coli has been determined. The operon consists of three open reading frames, glpABC, encoding polypeptides of molecular weight 62,000, 43,000, and 44,000, respectively. The 62,000- and 43,000-dalton subunits corresponded to the catalytic GlpAB dimer. The larger GlpA subunit contained a putative flavin adenine dinucleotide-binding site, and the smaller GlpB subunit contained a possible flavin mononucleotide-binding domain. The GlpC subunit contained two cysteine clusters typical of iron-sulfur-binding domains. This subunit was tightly associated with the envelope fraction and may function as the membrane anchor for the GlpAB dimer. Analysis of the GlpC primary structure indicated that the protein lacked extended hydrophobic sequences with the potential to form alpha-helices but did contain several long segments capable of forming transmembrane amphipathic helices.     

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli.

In Escherichia coli, the lct locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the lct locus contained three overlapping genes in the clockwise order of lctD (encoding a flavin mononucleotide-dependent dehydrogenase), lctR (encoding a putative regulator), and lctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A phi (lctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

A potential target enzyme for trypanocidal drugs revealed by the crystal structure of NAD-dependent glycerol-3-phosphate dehydrogenase from Leishmania mexicana

BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and L-glycerol-3-phosphate. Although the enzyme has been characterized and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid Trypanosoma brucei makes it a viable drug target against sleeping sickness. RESULTS: The 1.75 A crystal structure of apo GPDH from L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 A holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function. CONCLUSIONS: The crystal structure of GPDH from L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.     

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.