Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

The Formation of beta, 1 --> 4 Glucan from UDP-alpha-d-Glucose Catalyzed by a Phaseolus aureus Enzyme

Particulate enzyme preparations from Phaseolus aureus hypocotyls catalyze the formation of an alkali insoluble β, 1 → 4 linked [¹⁴C]-glucan using UDP-α-d [¹⁴C]-glucose as substrate. Particulate enzymes prepared from root tissue also catalyzed the production of β, 1 → 4 glucan. UDP-β-d-[¹⁴C]-glucose would not serve as a substrate for these enzymes. The presence or absence of β, 1 → 4 glucan synthetase activity was independent of tissue source, substrate concentration, or homogenization method.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [γ-³²P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg²⁺. ATP, and recovery of only [³²P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [γ-³²P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.     

A 3-aminobenzamide-resistant labeled protein in [P]NAD-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [³²P]NAD⁺. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [³²P]NAD⁺ was shown to be Mg²⁺-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [α-³²P]ATP, [γ-³²P]ATP and [³²P]orthophosphate, but not from [³H]NAD⁺ or [¹⁴C]NAD⁺. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [³²P]NAD⁺. This phosphorylation appears to occur via an Mg²⁺-dependent conversion of NAD⁺ to AMP with the eventual utilization of the α-phosphate for phosphorylation of the 72 000 molecular weight protein.     

A simple, rapid preparation ofα-[32P]- labelled adenosine diphosphate

Hexokinase (EC 2.7.1.1) will convert commercially available α-[³²P]-labelled ATP into α-[³²P]-labelled ADP. A simple, rapid isolation procedure for the α-[³²P]-labelled ADP is described and this synthetic method can be used for the preparation of other α-[³²P]-labelled nucleoside diphosphates.     

Enzymatic Synthesis and Characterization of Fructooligosaccharides and Novel Maltosylfructosides by Inulosucrase from Lactobacillus gasseri DSM 20604

The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter⁻¹) and 1.6 U ml⁻¹ of IS–25 mM sodium acetate buffer–1 mM CaCl₂ (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml⁻¹). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [β-d-fructofuranosyl-(2→6)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the β-2,1-linked fructose or the β-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors.     

Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls

An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 10⁴). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.     

In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-³²P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.     

Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination

The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of γ-³²P-ATP in the hexokinase reaction is outlined.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 μM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [γ-³²P]ATP and [γ-³²P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [γ-³²P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [γ-³²P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.     

Plant Pyruvate Dehydrogenase Complex: II. ATP-Dependent Inactivation and Phosphorylation

ATP inactivated plant pyruvate dehydrogenase complex (PDC) from broccoli (Brassica oleracea) mitochondria. ATP inactivation of the complex was time-dependent and proportional to the ATP concentration. Time-dependent incorporation of ³²P from [γ³²P]ATP into trichloroacetic acid-precipitable protein corresponded to the inactivation of the PDC. It is concluded that plant PDC is phosphorylated and inactivated by a PDC kinase.     

Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Etude du mechanisme de la regulation hormonale dans le placenta humain: Mise en evidence d'une activite adenyl cyclasique dans les homogenats de placenta a terme

Human full-term placentas possess adenyl cyclase activity which was assayed by the transformation of [α-³²P]ATP to cyclic 3′,5′-[³²P]AMP according to the method of Krishnaet al. (G. Krishna, B. Weiss and B. B. Brodie), J. Pharmacol. Exptl. Therap., 163 (1968) as modified by Rodbell (M. Rodbell, J. Biol. Chem., 242 (1967) 5744).     

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K_ATP channels, (SUR1/Kir6.2)₄, couple the transmembrane flux of K⁺, and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5′-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR1_E1507Q, with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5′-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[³²P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K_ATP channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.