Sn-glycerol 3-phosphate Dehydrogenase Research Articles

One-pot enzymatic reaction sequence for the syntheses of d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate

A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.

Novel Inhibitors of Mitochondrial sn-Glycerol 3-phosphate Dehydrogenase

Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and K i values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Preferential expression of cholesteryl ester transfer protein mRNA by stromal-vascular cells of human adipose tissue

Cholesteryl ester transfer protein (CETP) mRNA is more abundantly expressed in small mature adipocytes as compared to large, lipid-rich adipocytes [Radeau et al., J. Lipid Res. 36 (1995) 2552–2561]. In the present study, the stromal vascular fraction (SVF) of human adipose tissue was isolated and the presence of very small fat cells in this fraction confirmed by electron microscopy and by demonstrating the presence of mRNA for adipsin and for CCAAT enhancer binding protein α (C/EBPα), a marker of adipocyte differentiation. sn-Glycerol 3-phosphate dehydrogenase (GPDH) activity was present in the SVF but not in the preadipocyte fraction. Northern blot analysis of human adipose tissue demonstrated that CETP mRNA expression was significantly greater (+96%, P<0.03) in stromal-vascular cells (SVC) as compared to mature fat cells. By comparison, lipoprotein lipase mRNA expression was lower (−75%, P<0.03) in SVC while apolipoprotein E mRNA expression was not significantly different in SVC as compared to isolated adipocytes. By RT-PCR analysis, we demonstrated that CETP mRNA was expressed by human pre-adipocytes at levels less than those of SVC and adipocytes. The absence of monocytes/macrophages in SVC was confirmed by the absence of FcγRIII (CD16) mRNA in these fractions. These data demonstrate that CETP mRNA is most highly expressed in the immature fat cells of human adipose tissue, consistent with other experiments from this laboratory demonstrating that CETP plays an important local role in adipocyte cholesterol accumulation.

Ergosteroids II: Biologically Active Metabolites and Synthetic Derivatives of Dehydroepiandrosterone

An improved procedure for the synthesis of 3β-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17β-hydroxy derivative, or hydroxylated at 7α or 7β, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

Expression of gpsA encoding biosynthetic sn-glycerol 3-phosphate dehydrogenase suppresses both the LB- phenotype of a secB null mutant and the cold-sensitive phenotype of a secG null mutant.

SecB maintains the structures of a subset of precursor proteins competent for translocation across the Escherichia coli cytoplasmic membrane. SecG, a membrane component of the translocation machinery, stimulates protein translocation by undergoing the cycle of membrane topology inversion. Null mutants of secB and secG are unable to form isolated colonies on rich medium and at low temperature respectively. A 3.2 kb DNA fragment carrying the secB-gpsA region on a multicopy plasmid was found to suppress the null mutation of either gene. However, subcloning of the DNA fragment revealed that secB is not involved in the suppression of either mutation. Instead, gpsA located downstream from the secB gene was found to be responsible for the suppression of both mutations. The activity of the gpsA-encoded sn-glycerol-3-phosphate dehydrogenase, which is involved in phospholipid synthesis, was significantly lower in the secB null mutant than in the wild type, presumably because of a polar effect. Suppression of the secB null mutation required the wild-type level of GpsA activity. In contrast, overexpression of the enzyme was essential for suppression of the secG null mutation. Moreover, the gpsA-dependent suppression of the secG null mutation occurred only on rich medium, i.e. not on minimal medium. These results indicate that the SecB function is dispensable even in rich medium, and further demonstrate that overexpression of enzymes involved in phospholipid synthesis partly compensates for the SecG function.

Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12.

Aerobic sn-glycerol 3-phosphate dehydrogenase is a cytoplasmic membrane-associated respiratory enzyme encoded by the glpD gene of Escherichia coli. The glpD operon is tightly controlled by cooperative binding of the glp repressor to tandem operators (O(D)1 and O(D)2) that cover the -10 promoter element and 30 bp downstream of the transcription start site. In this work, two additional operators were identified within the glpD structural gene at positions 568 to 587 (0(D)3) and 609 to 628 (0(D)4). The two internal operators bound the glp repressor in the presence or absence of the tandem operators (O(D)1 and O(D)2) in vitro, as shown by DNase I footprinting. To assess a potential regulatory role for the two internal operators in vivo, a glpD-lacZ transcriptional fusion containing all four operators was constructed. The response of this fusion to the glp repressor was compared with those of fusion constructs in which O(D)3 and O(D)4 were inactivated by either deletion or site-directed mutagenesis. It was found that the repression conferred by binding of the glp repressor to O(D)1 and O(D)2 was increased five- to sevenfold upon introduction of the internal operators. A regulatory role for HU was suggested when it was found that repressor-mediated control of glpD transcription was increased fourfold in strains containing HU compared with that of strains deficient in HU. The effect of HU was apparent only in the presence of all four glpD operators. The results suggest that glpD is controlled by formation of a repression loop between the tandem and internal operators. HU may assist repression by bending the DNA to facilitate loop formation.

Cloning and characterization of GPD2, a second gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino-terminal extension of unknown function. GPD2 is located on chromosome VII and cross-hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild-type cells. Analysis of gene transcription by a CAT-reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on nonfermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8-17-fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4 M NaCl.

Beta-lactam-induced bacteriolysis of amino acid-deprived Escherichia coli is dependent on phospholipid synthesis

The penicillin tolerance of amino acid-deprived relA+ Escherichia coli is attributed to the stringent response; i.e., relaxation of the stringent response suppresses penicillin tolerance. The beta-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic sn-glycerol 3-phosphate dehydrogenase). Therefore, beta-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. The lysis process during amino acid deprivation can be experimentally dissociated into two stages designated the priming stage (during which the interaction between the beta-lactam and the penicillin-binding proteins occurs) and the beta-lactam-independent lysis induction stage. Both stages were shown to require phospholipid synthesis. It has been known for some time that the inhibition of phospholipid synthesis is among the plethora of physiological changes resulting from the stringent response. These results indicate that the inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response are both secondary consequences of the inhibition of phospholipid synthesis.

A gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae.

Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive glycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1-osg4). The osg1-1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD(+)-dependent sn-glycerol 3-phosphate dehydrogenase (GPD), an enzyme in the glycerol-producing pathway. Complementation of the osg1-1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47-50% identity with GPD from other sources. Micro-sequencing of the N-terminus of purified S. cerevisiae GPD revealed a 20-amino-acid sequence that was identical to a nucleotide-deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N-terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre-sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.

Induction of Hepatic Mitochondrial Glycerophosphate Dehydrogenase in Rats by Dehydroepiandrosterone1

Feeding the thermogenic steroid, 5-androsten-3 beta-ol-17-one (dehydroepiandrosterone, DHEA) in the diet of rats induced the synthesis of liver mitochondrial sn-glycerol 3-phosphate dehydrogenase to levels three to five times that of control rats within 7 days. The previously reported enhancement of liver cytosolic malic enzyme was confirmed. The induction of both enzymes was detectable at 0.01% DHEA in the diet, reached plateau stimulation at 0.1 to 0.2%, and was completely blocked by simultaneous treatment with actinomycin D. Feeding DHEA caused smaller, but statistically significant increases of liver cytosolic lactate, sn-glycerol 3-phosphate, and isocitrate (NADP(+)-linked) dehydrogenases but not of malate or glucose 6-phosphate dehydrogenases. The capability of DHEA to enhance mitochondrial glycerophosphate dehydrogenase and malic enzyme was influenced by the thyroid status of the rats; was smallest in thyroidectomized rats and highest in rats treated with triiodothyronine. 5-Androsten-3 beta,17 beta-diol and 5-androsten-3 beta-ol-7,17-dione were as effective as DHEA in enhancing the liver mitochondrial glycerophosphate dehydrogenase and malic enzyme. Administering compounds that induce the formation of cytochrome P450 enzymes enhanced liver malic enzyme activity but not that of mitochondrial glycerophosphate dehydrogenase. Arochlor 1254 and 3-methylcholanthrene also increased the response of malic enzyme to DHEA feeding.

Glycerol catabolism in Aspergillus nidulans

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol 3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction. Biochemical and genetical analyses show that dihydroxyacetone and D-glyceraldehyde are converted into glycerol and then catabolized by the same pathway. D-Glyceraldehyde can be reduced by NADP(+)-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.

Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Aerobic sn-glycerol 3-phosphate dehydrogenase, encoded by the glpD gene of Escherichia coli, is a cytoplasmic membrane-associated respiratory enzyme. The nucleotide sequence of glpD was determined. An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence. The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a glpD-lacZ translational fusion. The predicted molecular weight, 56,750, corresponds well with the reported value of 58,000 for purified sn-glycerol 3-phosphate dehydrogenase. The flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins from E. coli. Repetitive extragenic palindromic sequences were identified downstream of the glpD coding region. The site for transcription termination was located between 87 and 216 bp downstream of the translation stop codon.

Microcalorimetric monitoring of growth of Saccharomyces cerevisiae: osmotolerance in relation to physiological state.

The importance of the physiological state of a culture of Saccharomyces cerevisiae for tolerance to sudden osmotic dehydration was studied, and it was investigated whether specific osmotolerance factors were demonstrable. The microcalorimeter was used to monitor growth, and different physiological states of the culture were selected and their osmotolerance was tested. In addition to cells in the stationary phase, cells from the transition phase between respirofermentative and respiratory catabolism were osmotolerant. S. cerevisiae exhibited ever-changing metabolism during batch growth on either glucose or ethanol as the carbon source. Instantaneous heat production per biomass formation (dQ/dX) and specific activity of sn-glycerol 3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) were shown to differ for different physiological states. Neither high respiratory activity nor low total cellular activity, nor factors involved in osmoregulation, i.e., intracellular glycerol or activity of GPDH, correlated with the osmotolerant phenotype.

Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii.

A glycerol-nonutilizing mutant of the salt-tolerant yeast Debaryomyces hansenii was isolated. When subjected to salt stress the mutant produced glycerol, and the internal level of glycerol increased linearly in proportion to increases of external salinity as in the wild-type strain. However, at increased salinity the mutant showed a more pronounced decrease of growth rate and growth yield and lost more glycerol to the surrounding medium than did the wild type. Uptake experiments showed glycerol to be accumulated against a strong concentration gradient, and both strains displayed similar kinetic parameters for the uptake of glycerol. An examination of enzyme activities of the glycerol metabolism revealed that the apparent Km of the sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5) was increased 330-fold for sn-glycerol 3-phosphate in the mutant. Based on the findings, a scheme for the pathways of glycerol metabolism is suggested.

ESTIMATION OF THE RATE OF GLYCEROL 3-PHOSPHATE DEHYDROGENASE EVOLUTION IN HIGHER VERTEBRATES.

Among proteins which have been examined for polymorphism in natural populations, sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) is one of the least polymorphic (Ayala et al., 1974; Collier and Maclntyre, 1977, 1978; Lakovaara et al., 1977; Coyne et al., 1979; Johnson, 1979). These studies have also indicated that the enzyme, also known as aXGPDH, is evolving rather slowly. It is possible that limited polymorphism is correlated with slow rates of amino acid replacement (Skibinski and Ward, 1982); however, for most enzymes whose natural polymorphism has been well-studied there are very few amino acid sequence data. Such is the case for glycerol 3-phosphate dehydrogenase. The only published sequence is for the rabbit muscle enzyme (Otto et al., 1977, 1980). The structure of the Drosophila enzyme is under study (Niesel et al., 1982). Glycerol 3-phosphate dehydrogenase is a cytosolic enzyme present in most tissues of all eukaryotes. In vertebrates amounts of this enzyme are particularly high in liver and white skeletal muscle, but relatively low in heart, brain, and most neoplastic tissues (Young and Pace, 1958). This enzyme is particularly concentrated in the flight muscles of insects such as bees and flies (Crabtree and Newsholme, 1972) where the enzyme

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.

Bis(monoacylglycero)phosphate in alkalophilic bacteria.

Bis(monoacylglycero)phosphate (BMP) was found in an alkalophilic bacterium, Bacillus sp. A-007. This lipid comprised 4% of the phospholipid of this organism. The very low rate and extent of triphenylmethylation of BMP indicated that the two fatty acid residues occupied the C-1 and C-3' positions of the two glycerol moieties. The stereochemical configuration of the backbone of BMP was identified as sn-glycero-3-phosphoryl-1'-sn-glycerol by sn-glycerol 3-phosphate dehydrogenase analysis after acetic acid hydrolysis and mild alkaline methanolysis. This configuration was the same as that of phosphatidylglycerol, but differed from that of BMP from mammalian cells. Finally, the structure of BMP in Bacillus sp. A-007 was concluded to be 1-acyl-sn-glycero-3-phosphoryl-1' (3'-acyl)-sn-glycerol. The fatty acids of BMP were those common in Bacillus species. The fatty acid composition of BMP was very similar to that of the major lipids in this organism. The fatty acid distribution between the C-1 and C-3' positions of BMP was determined by a newly developed method which included successive steps of acetic acid hydrolysis, chemical acylation and phospholipase A2 treatment. Fatty acids with longer chain length were concentrated in the C-1 position, and the shorter fatty acids were the major ones esterified at the C-3' position.

Caulobacter cresentus mutant defective in membrane phospholipid synthesis.

To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.

Resolution of glpA and glpT loci into separate operons in Escherichia coli K-12 strains

The independent insertion of bacteriophage Mu into the gene coding for anaerobic sn-glycerol 3-phosphate dehydrogenase (glpA) or into the genes coding for sn-glycerol 3-phosphate transport (glpT) suggested that these two closely linked loci are in separate operons.