Anomeric specificity of glucose effect on cAMP, fructose 1,6-bisphosphatase, and trehalase in yeast

The addition of beta-D-glucose (final concentration, 50 mM) to a cell suspension of Saccharomyces cerevisiae in stationary phase caused a rapid 4-fold increase in the concentration of cAMP, while a 2-fold increase of cAMP was observed by the addition of alpha-D-glucose. beta -D-Glucose was also more effective than alpha-D-glucose in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase. These results, taken together with the previous report that alpha-D-glucose is transported more rapidly than beta-D-glucose in Saccharomyces cerevisiae, do not support the view currently proposed by some investigators that cotransport of D-glucose with protons causes the depolarization of the cell membrane, resulting in the activation of adenylate cyclase. The present data, however, provides supporting evidence for the view that cAMP-dependent protein kinase is implicated in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase.     

Arginine modification with butanedione inhibits the potassium ATPase of Streptococcus faecalis

The K+-ATPase of Streptococcus faecalis is inhibited by incubation with the arginine-modifying reagent 2,3-butanedione. The inactivation proceeds by pseudo - first order kinetics and a double-logarithmic plot of the pseudo - first order rate constants versus reagent concentrations yields a reaction order of 1.14 with respect to butanedione. Partially inactivated ATPase exhibits a decreased maximal velocity but the same affinity for ATP, as compared to the native enzyme. Butanedione modification is inhibited by adenine nucleotides. These results indicate the involvement of most likely one crucial arginyl residue in adenine nucleotide binding by the ATPase.     

Simultaneous measurement of atrial natriuretic polypeptide (ANP) messenger RNA and ANP in rat heart--evidence for a preferentially increased synthesis and secretion of ANP in left atrium of spontaneously hypertensive rats (SHR)

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.     

The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.     

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.     

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.     

A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyl-lysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01-100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activators is demonstrated.     

Fibrinolysis by urokinase endowed with magnetic property

The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.     

Toxicity of enzymically-oxidized low-density lipoprotein

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.     

Internal anion binding site and membrane potential dominate the regulation of proton pumping by the chromaffin granule ATPase

Effects of anions and membrane potential on the reconstituted proton pump from chromaffin granules were investigated. When acetate was present inside of the vesicles, ATP-dependent proton uptake was absolutely dependent on external chloride. Without external chloride, however, substantial proton uptake was observed when chloride or sulfate was present inside of the vesicles. Inside negative membrane potential drove ATP-dependent proton uptake regardless of the anion species present inside or outside of the vesicles. It is concluded that the internal anion binding site and membrane potential regulate the proton pumping activity of the ATPase.