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Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.
A recent study from this laboratory (G-q. Yu, D. Goldrick, H.R. Kaback and J-s. Hong, in preparation) indicates that the externally induced phosphoglycerate transport system (pgt) of Salmonella typhimurium is positively regulated by the activator gene, pgtA, and that the pgtA is localized in the SalI-PstI restriction fragment 3.0 kb from the permease gene, pgtP. In this paper, we describe the identification of the activator gene and its gene product and the determination of the complete nucleotide (nt) sequence of the activator gene as well as of a downstream gene not required for pgtP expression. The amino acid sequence of the activator based on the nt sequence shows an N-terminal signal-like sequence which is apparently not cleaved and three potential transmembrane sequences in the C-terminal half of the protein based on the hydropathy analysis.
The conversion of D-lactate to pyruvate in isolated membrane preparations of E. coli ML 308-225 markedly stimulates the transport of proline, glutamic acid, aspartic acid, aspargine, tryptophan, lysine, serine, alanine, and glycine. The uptake of histidine, phenylalanine, tyrosine, leucine, isoleucine, and valine by the membranes is also markedly stimulated by this conversion, although these amino acids are taken up much less effectively than those mentioned previously. The uptake of arginine, methionine, cystine, and cysteine is enhanced only about twofold in the presence of D-(-)-lactate, and these amino acids are not concentrated well by the membranes. With the exception of glutamate, asparate, asparagine, and methionine, which are converted to other metabolites to varying extents in the intramembranal pool, each of the other amino acids was recovered from the membranes as the unchanged amino acid. Succinate, L-(+)-lactate, D,L-alpha-hydroxybutyrate, and DPNH partially replace D-(-)-lactate but are less effective.
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We identified the phosphoglycerate transporter gene of Salmonella typhimurium and its polypeptide product and determined the nucleotide sequence of the gene. The predicted translation product was a protein of 406 amino acid residues and was extremely hydrophobic, a feature that is consistent with its role in membrane transport. Hydropathy analysis suggested that there are eight transmembrane segments of at least 20 amino acid residues for the protein. The transcription start point was mapped to lie at position -44 relative to the putative translational initiation start point. Comparison of PgtP with UhpT and GlpT, the membrane-bound proteins involved in the transport of hexose-6-phosphate and glycerol-3-phosphate, respectively, revealed a very high degree of amino acid sequence similarity among them, reflecting not only similar structures and functions among these polypeptides but also a common evolutionary origin for them.
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ADP-glucose pyrophosphorylase from photosynthetic tissue is allosterically regulated by 3-phosphoglycerate and inorganic phosphate. In contrast, data from our laboratory indicated that the major AGPase from barley seeds is insensitive to these regulators. Verification of this conclusion has, however, been hindered by the proteolytic degradation of the enzyme from seeds. This report characterizes the barley seed AGPase expressed in the baculovirus-insect cell system, confirming that lack of allosteric regulation by 3-PGA/Pi is an intrinsic property of the enzyme. Purification of the enzyme was by Ni2+-NTA agarose chromatography using a (His)6 tag attached to the N-terminus of the small AGPase subunit.
A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.
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Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.