Phos

Abstract: Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunological techniques, we have compared the synthesis of the phoA protein (alkaline phosphatase) and the phoS protein (phosphate-binding protein) in response to the level of phosphate in the medium in different genetic backgrounds containing the known alkaline phosphatase control mutations. Both proteins are produced in excess phosphate media in a phoR1a- strain, whereas neither protein is produced in a phoB- strain even under derepression conditions. In four different phoR1c- strains, however, the phoA product cannot be detected in extracts of cells obtained from any growth condition, whereas the phoS product is produced in both excess and limiting phosphate media. It is not yet known if phoR1c- mutants are a special class of mutations within the phoB gene or whether they occur in a separate cistron involved in alkaline phosphatase regulation. From these results we conclude that the expression of the phoA gene is not always co-regulated with expression of the phoS gene product. We have determined that the phoS protein is a component of periplasmic protein band P4 described by Morris et al. (1974). The phoS product lacks sulfur-containing amino acids and is extractable by treatment with polymyxin sulfate. The other component of band P4 contains methionine and/or cysteine and is not extracted by polymyxin sulfate treatment. Like the phoS and phoA proteins, its synthesis is sensitive to the concentration of phosphate in the growth medium. In addition, the existence of a new class of periplasmic proteins synthesized at maximum rate in high phosphate media is demonstrated.

Abstract: phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.

Abstract: This study examined muscle glycogenolysis and the regulation of glycogen phosphorylase (Phos) activity during 15 min of cycling at 85% of maximal O2 consumption (VO2max) in control and high free fatty acid (FFA; Intralipid-heparin) conditions in 11 subjects. Muscle biopsies were sampled at rest and 1, 5, and 15 min of exercise, and glycogen Phos transformation state (%Phos alpha), substrate (Pi, glycogen), and allosteric regulator (ADP, AMP, IMP) contents were measured. Infusion of intralipid elevated plasma FFA from 0.32 +/- 0.04 mM at rest to 1.00 +/- 0.04 mM just before exercise and 1.12 +/- 0.10 mM at 14 min of exercise. In the control trial, plasma FFA were 0.36 +/- 0.04 mM at rest and unchanged at the end of exercise (0.34 +/- 0.03 mM). Seven subjects used less muscle glycogen (46.7 +/- 7.6%, mean +/- SE) during the Intralipid trial, and four did not respond. In subjects who spared glycogen, glycogen Phos transformation into the active (alpha) form was unaffected by high FFA except for a nonsignificant reduction during the initial 5 min of exercise. Total AMP and IMP contents were not significantly different during exercise between trials, but total ADP was significantly lower with Intralipid only at 15 min. The calculated free ADP, AMP, and Pi contents were lower with Intralipid but not significantly different. However, when the present results were pooled with the data from a previous study using the same protocol [Dyck et al., Am. J. Physiol. 265 (Endocrinol, Metab. 28): E852-E859, 1993], the free ADP, AMP, and Pi contents of all subjects who spared glycogen (n = 13) were significantly lower at 15 min in the Intralipid trial. The findings suggest that the elevation of plasma FFA during intense cycling spares muscle glycogen by posttransformational regulation of Phos. This may be due to blunted increases in the contents of AMP, an allosteric activator of Phos alpha, and Pi, a substrate for Phos.