Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function.

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).     

Acquisitive evolution of ribitol dehydrogenase in Klebsiella pneumoniae.

Selection in continuous culture of Klebsiella pneumoniae mutants that have gained the ability to utilize xylitol while also retaining regulatory control over ribitol utilization was achieved with a dual-substrate regime. Initial steady-state cultures of wild-type organisms were maintained with 0.005% (0.329 mM) ribitol. Mutants of various types proliferated when the composition of the limiting medium was changed to 0.005% ribitol plus 0.250% (16.43 mM) xylitol.     

Paper replication method for isolation of radiation-sensitive mutants.

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.     

Age-related and seasonal variation in the Sertoli cell population, daily sperm production and serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone in stallions

Testes and blood samples were obtained from 201 stallions aged 6 months to 20 years in either December-January (nonbreeding season) or June-July (breeding season) to study the effect of age and season on reproductive parameters. Seasonal differences in the Sertoli cell population of adult (4-20 years old) horses were characterized by a 36% larger number of Sertoli cells in the breeding season than in the nonbreeding season. Seasonal elevation in the Sertoli cell population was associated with an increase in testicular weight and daily sperm production per testis (DSP/testis). Concentrations of luteinizing hormone (LH) and testosterone in serum varied with season. Although follicle-stimulating hormone (FSH) concentrations also tended to be higher in the breeding season, this trend was not statistically significant (P less than 0.08). Sertoli cell numbers averaged over both seasons, like testicular weights, increased with age until 4-5 years of age, but were stabilized thereafter. This age-related difference was also associated with increased concentrations of FSH, LH and testosterone, and with increased DSP/testis. The Sertoli cell population was capable of increasing in the adult horse by fluctuating its size with season. The number of elongated spermatids per Sertoli cell over both seasons increased with age up to 4-5 years of age and was stabilized thereafter. Thus, seasonal and/or age-related differences in DSP/testis were associated with significant elevations in serum concentrations of FSH, LH and testosterone, testicular weights, numbers of elongated spermatids per Sertoli cell and elevation of the Sertoli cell population.     

The transsynaptic regulation of the septal-hippocampal cholinergic neurons

There is not yet a complete understanding of the functional interactions among various septal nuclei which regulate hippocampal function. Nevertheless, much has been learned histologically and biochemically about the major connections of the distinct areas of the septal complex and the chemical character of some of these pathways. The cholinergic septal-hippocampal pathway serves as a well defined link between these two important structures of the limbic system. Acetylcholine turnover rates in the hippocampus have been shown to increase or decrease proportionally to the activity of the cholinergic neurons originating in the septum. Moreover, these turnover rates have been shown to be modulated by intraseptal injections of agonists or antagonists of various neurotransmitters or neuromodulators which are stored in various cell groups located in the septum. By coupling this biochemical approach with techniques to study the receptor organization, greater detail concerning the transmitter and cotransmitter interactions among the various neuromodulators can be obtained.     

Feedback inhibition of homoserine kinase from radish leaves

Homoserine kinase is a potential control point in the biosynthetic pathway for threonine, isoleucine and methionine. The radish leaf enzyme was tested     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.