Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.     

No first night shift effect observed following a nocturnal main sleep and a prophylactic 1-h afternoon nap

ABSTRACT

Neurobehavioural impairment on the first night shift is often greater than on subsequent night shifts due to extended wakefulness. The aim of the study was to determine whether a 1-h afternoon nap prior to the first night shift is sufficient to produce neurobehavioural performance at levels comparable to the second night shift. Twelve male volunteers (mean age 22.9 years) participated in a laboratory protocol that simulated two 12-h night shifts. A nap preceded the first shift and a 7-h daytime sleep was scheduled between shifts. Neurobehavioural performance and subjective sleepiness measured across each night did not significantly differ between first and second shifts.     

Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose hisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

The pharmacology of prazosin, a novel antihypertensive agent

During the past few years a large amount of pharmacological and physiological evidence has been obtained in favor of two distinct types of α-adrenoceptors. As a working hypothesis, it is feasible to assume that both α₁- and α₂-adrenoceptors are abundant on the vascular effector site, whereas the α-adrenoceptors (the blockade of which increases norepinephrine release) predominate at the level of peripheral sympathetic nerve endings. Prazosin is a novel, selective antagonist of α₁-adrenoceptors and can be considered an important advancement both pharmacologically and therapeutically since this compound in contrast to classical α-adrenoceptor blocking agents, is effective for the treatment of high blood pressure. Prazosin lacks direct myorelaxant properties and, unlike many vasodilators, in doses lowering blood pressure it does not produce undesirable increases in heart rate and plasma renin activity. Prazosin has proved to be a very useful pharmacological tool since it has permitted us the furtherance of our knowledge with respect to the subclassification of receptors mediating the effects produced by α-adrenoceptor agonists, particularly clonidine. Pharmacokinetic and metabolic studies on prazosin given orally indicate that in animals and in man this compound has a low bioavailability, short half life and undergoes extensive biotransformation. The most common clinical use of prazosin is as an antihypertensive agent and is often given in association with established blood pressure lowering drugs. Recently, it was shown to be useful in the treatment of congestive heart failure, but for this application tolerance has been described. Generally, patients treated chronically with prazosin suffer only minor unwanted effects. This is in contrast to past experience with traditional α-adrenoceptor antagonist. The most serious side effect of prazosin is known as the “first dose phenomenon” which can sometimes lead to syncope. However, it can be avoided if prazosin therapy is initiated with minimally effective doses and individually tailored to obtain the desired antihypertensive effect. Presently, the interesting clinical profile of prazosin is attributed to its novel property of being a selective antagonist of postsynaptic α₁-adrenoceptors. Howeverm this is probably an over simplification since some therapeutic observations are not entirely consistent with results which would have been expected for a selective α₁-adrenoceptor. For example, prazosin, like the classical antagonists, would be expected to produce sexual dysfunction but, in fact, does not to any significant degree. Future studies with new chemical structures sharing the pharmacological profile of prazosin will clarify the real role of the selectivity towards α₁-adrenoceptors in the therapeutic success of prazosin.     

SHOOT HISTOGENESIS: SUBAPICAL MERISTEMATIC ACTIVITY IN A CAULESCENT PLANT AND THE ACTION OF GIBBERELLIC ACID AND AMO-1618

Sachs , R. M., A. Lang , C. F. Bretz and Joan Roach . (U. California, Los Angeles.) Shoot histogenesis: subapical meristematic activity in a caulescent plant and the action of gibberellic acid and Amo—1618. Amer. Jour. Bot. 47(4): 260—266. Illus. 1960.–Studies on gibbereilininduced stem formation in rosette plants (Sachs et al., 1959) have shown that a zone of intensive meristematic activity, arising below the existing apical meristem, is almost solely responsible for stem histogenesis, i.e., the formation of the cells constituting the elongate stem. An extensive subapical zone of meristematic activity is also present in caulescent plants, such as Chrysanthemum morifolium, Amo-1618 ([4-hydroxy-5 isopropyl-2 methylphenyl] trimethylammonium chloride, 1-piperidine carboxylate) completely inhibits subapical meristematic activity in chrysanthemum, causing the plants to assume a dwarfed, rosette-like habit of growth. Gibberellic acid, applied either simultaneously, or following the Amo—1618 treatment, completely prevents or reverses the effect of Amo—1618, making the plants retain or resume their normal growth habit. Amo—1618 and gibberellic acid have relatively little effect upon the activity of the apical meristem of Chrysanthemum. Thus, while the apical meristem proper (eu- or promeristem) is the site of shoot organization and the ultimate source of the cells of the entire shoot, the subapical zone of division, termed the subapical meristem, is largely responsible for stem histogenesis in caulescent as well as in rosette plants. Gibberellins, or native, gibberellin-like substances appear to regulate the activity of the subapical meristem and thus to play an important role in shoot development. Amo—1618 and related compounds seem to exert their dwarfing effect in plants by acting as antagonists of gibberellins, at least with respect to the latters' function in regulating the subapical meristematic activity in the shoot.     

Multiple phosphorylation of mouse muscle glycogen synthase

Glycogen synthase was isolated from extracts of mouse diaphragm muscle by immunoprecipitation with specific antibodies raised against the rabbit muscle enzyme. A procedure was developed which permitted phosphorylation of the immunoprecipitated enzyme by several purified protein kinases. Peptide mapping techniques (including reverse-phase HPLC and thin-layer electrophoresis and chromatography) were used to compare tryptic phosphopeptides of the rabbit and mouse muscle enzymes. The results demonstrated a high degree of similarity in the chemical properties of these peptides, suggesting significant homology around the phosphorylation sites in these proteins. Thus, mouse peptides corresponding to the rabbit muscle peptides containing sites 1a, 1b, 2, 3, and 5 were identified, with protein kinase recognition specificities identical to those of the rabbit enzyme. The study indicates significant conservation in the muscle isozymes of glycogen synthase between mouse and rabbit as well as a similar distribution of phosphorylation sites throughout the enzyme subunit.     

Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca²⁺ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.