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J Preiss

Publications and source records attributed to J Preiss.

At least 181 records · Page 10Linked to original sources
PubMed1980

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa.

The ADPglucose pyrophosphorylases from Rhodopseudomonas sphaeroides and Rhodopseudomonas gelatinosa are activated by fructose-6-phosphate, pyruvate and fructose-1,6 biophosphate-P2. The effects of the activators are to increase significantly the Vmax of ADPglucose synthesis and to lower the S0.5 values (concentration of substrates giving 50% maximal velocity) for ATP and MgCl2. The R. sphaeroides enzyme is inhibited by Pi while the R. gelatinosa enzyme is inhibited by AMP as well as by Pi. The interaction between inhibitor and activator is complex. At very low concentrations of activator the enzyme is more sensitized to inhibition. However, at higher concentrations of activator there is a decrease in the sensitivity of the enzyme towards inhibition. The findings are discussed with respect to glycogen synthesis in these microorganisms and may be related to findings that indicate that Rhodopseudomonads have the ability to degrade sugars via the Entner-Duodoroff or Embden-Meyerhoff pathways.

Adenosine Diphosphate Glucose↗
PubMed1980

Characterization of the spinach leaf phosphorylases.

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V(max) for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K(m) for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K(m) values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V(max) values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K(m) values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K(m) value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity.

Journal Article↗
PubMed1980

Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES.

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, beta-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca(2+) requirement or heat stability of seed endosperm alpha-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.The chloroplastic R-enzyme debranches amylopectin, beta-limit amylopectin, pullulan, and alpha-limit dextrins, but not rabbit liver glycogen. An increase in extinction coefficient and lambda(max) is detected when the debranched amylopectin and beta-limit amylopectin form a complex with I(2)-KI. Based on these properties, the chloroplastic R-enzyme is similar in enzymic activity to the R-enzyme observed in endosperm tissue.

Journal Article↗
PubMed1980

Biosynthesis of bacterial glycogen: purification and properties of Salmonella typhimurium LT-2 adenosine diphosphate glucose pyrophosphorylase.

The adenosine diphosphate glucose pyrophosphorylase from a Salmonella typhimurium LT-2 mutant, JP102, derepressed in the glycogen biosynthetic enzymes was purified to homogeneity. The enzyme was found to be identical with the parent wild-type enzyme with respect to regulatory properties, immunological reactivity, and kinetic constants for the allosteric effectors and for the substrate, adenosine triphosphate. The JP102 enzyme was composed of four identical subunits, each with a molecular weight of about 48,000. This was supported by the findings that (i) gel electrophoresis under denaturing conditions showed only one component; (ii) digestion with carboxypeptidase B released stoichiometric amounts of arginine, and (iii) amino-terminal sequencing showed a single sequence for the first 27 residues. The properties of the purified S. typhimurium enzyme were compared with the properties of the previously purified Escherichia coli B enzyme.

Adenosine Diphosphate Glucose↗
PubMed1979

Biosynthesis of bacterial glycogen. The nature of the binding of substrates and effectors to ADP-glucose synthase.

Kinetic studies with ADP-glucose synthase show that 1,6-hexanediol bisphosphate (1,6-hexanediol-P2) is an effective activator that causes the enzyme to have a higher apparent affinity for ATP- and ADP-glucose than when fructose-1,6-P2 is the activator. Furthermore, in the presence of 1,6-hexanediol-P2, substrate saturation curves are hyperbolic shaped rather than sigmoidal shaped. CrATP behaves like a nonreactive analogue of ATP. Kinetic studies show that it is competitive with ATP. CrATP is not a competitive inhibitor of ADP-glucose. However, the combined addition of CrATP and glucose-1-P inhibits the enzyme competitively when ADP-glucose is the substrate. In binding experiments, CrATP, ATP, and fructose-P2 appear to bind to only half of the expected sites in the tetrameric enzyme, while ADP-glucose, the activators, pyridoxal-P and 1,6-hexanediol-P2, and the inhibitor, AMP, bind to four sites/tetrameric enzyme. Fructose-P2 inhibits 1,6-hexanediol-P2 binding, suggesting competition for the same sites. Glucose-1-P does not bind to the enzyme unless MgCl2 and CrATP are present and binds to four sites/tetrameric enzyme. Alternatively, CrATP in the presence of glucose-1-P binds to four sites/tetrameric enzyme. Thus, there are binding sites for the substrates, activators, and inhibitor located on each subunit and the binding sites can interact homotropically and heterotropically. ATP and fructose-P2 binding is synergistic showing heterotropic cooperativity. ATP and fructose-P2 must also be present together to effectively inhibit AMP binding. A mechanism is proposed which explains some of the kinetic and binding properties in terms of an asymmetry in the distribution of the conformational states of the four identical subunits.

Adenosine Diphosphate Glucose↗
PubMed1979

Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves.

The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chloroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADP-Glucose pyrophosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosylase) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction. Chromatography of a chloroplast extract on diethylaminoethyl-cellulose resolves the R- and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amylopectin as a substrate indicates an endolytic activity but displays properties unlike the typical alpha-amylase as isolated from endosperm tissue.

Journal Article↗
PubMed1979

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels.

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K(m) for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.

Journal Article↗
PubMed1978

Biosynthesis of bacterial glycogen. Isolation and characterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of the allosteric effector, 1,6-hexanediol-P2, protects against pyridoxylation of the allosteric activator site and allows modification of the ADP-glucose-protected site. The activator site CNBr [3H]pyridoxyl-P peptide was purified to homogeneity in the presence of urea by Sephadex G-50 and CM-cellulose chromatography. The peptide consists of 59 residues, with a molecular weight of 6750. The NH2-terminal of the peptide has a 16-residue sequence overlap with the previously determined NH2-terminal sequence of the native enzyme. The activator site pyridoxyl-P lysine is identified as residue 38 of the native enzyme's NH2 terminus. The ADP-glucose-protected site CNBr [3H]pyridoxyl peptide was purified to homogeneity by Sephadex G-50 and DEAE-cellulose chromatography. The peptide consists of 21 residues, with a molecular weight of 2460. The sequence of this peptide has been elucidated.

Adenosine Diphosphate Glucose↗
PubMed1978

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.

Adenosine Diphosphate Glucose↗
PubMed1978

Amylopectin degradation in pea chloroplast extracts.

Phosphorolysis rather than phosphorylation of amylolysis products was found to be the major pathway of sugar phosphate formation from amylopectin by pea (Pisum sativum L.) chloroplast stromal proteins. The K(m) for inorganic phosphate incorporation was 2.5 mm, and ATP did not stimulate amylopectin-dependent phosphate incorporation. Arsenate (10 mm) inhibited phosphate incorporation into glucose monophosphates up to 46% and phosphoglucomutase activity 96%, resulting in glucose 1-phosphate accumulation as a product of amylopectin degradation. The intracellular distribution of enzymes of starch utilization was determined. Phosphorylase, phosphoglucomutase, and hexokinase were found in the chloroplast and cytoplasm, while beta-amylase was restricted to the cytoplasm. Maltase was not detectable; maltose phosphorylase was active in the chloroplast.

Journal Article↗
PubMed1978

Purification and properties of sucrose synthase from maize kernels.

Sucrose synthase was purified from 22-day-old maize (Zea mays L.) kernels to homogeneity by the successive steps of ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and affinity chromatography on a UDP-hexanol-amino-agarose column. The degree of purification is 42-fold and the yield is over 80%. Polyacrylamide gel electrophoretic techniques, sedimentation velocity, and gel filtration studies revealed that the enzyme has identical subunits and could assume tetrameric, octameric, and other higher aggregated forms which are dependent on the ionic species and ionic strength of the solution. All of the enzyme forms exhibit catalytic activity but show differences in their specific activities. In most cases, the tetramer is the predominant form and has the highest specific activity. It is thus concluded that the tetramer could be the native form of the enzyme. The subunit protein has a molecular weight of 88,000 and a blocked NH(2) terminus which is not available to Edman degradation. Some general properties and the amino acid composition of the enzyme are also reported.

Journal Article↗