Phosphotransfer reactions as a means of G protein activation

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound subunits and free dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTR The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews). (Mol Cell Biochem 157: 593, 1996)Recipient of Servier Investigator Award     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

Total, organic and extractable-P in humus and soil beneath Sitka spruce planted in pure stands and in mixture with Scots pine

Total, organic and extractable P were measured in the humus and underlying soil to 10 cm depth beneath Sitka spruce (SS) and mixed Sitka spruce and Scots pine (SS+SP) stands planted on upland heath. The humus beneath SS+SP contained significantly (p<0.01) greater amounts of total and organic-P than that in SS and the mixed stands had more effectively retained approximately 87 per cent of previously applied fertilizer-P, totalling 100 kg P ha^–1, compared with 70 per cent in SS. Despite the larger amounts of total-P in the mixed plots 0.01 M CaCl₂ extractable molybdate reactive phosphorus (MRP) was significantly (p<0.05) greater in SS+SP humus only during March and April. Greater concentrations of MRP were released from the humus and soil during July and August at a mean rate of 58 g P ha^–1 day^–1. This coincided with drying of the soil during the summer and the rate of release, attributed to death of fine roots and microorganisms, was 4 to 30 times greater than reported values for rates of net mineralization of P from forest soils.     

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned fromDrosophila melanogaster and one is fromArtemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.     

Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

Kinetics of luxury uptake of phosphate by algae-dominated benthic communities

The uptake of phosphate by benthic communities, dominated by living algae, previously exposed to different levels of external nutrient loading, exhibited first-order kinetics with respect to the intracellular P-deficit. This deficit is the difference between the maximum and the actual intracellular P-concentration.The maximum storage capacity of P per unit of dry weight was positively correlated to the level of external nutrient loading, whereas the phosphate uptake rate constant was negatively correlated.The observed internal P concentrations in the benthic layer of test ditches over a period of two and a half years, indicated a slight decrease towards a minimum value in a ditch with a low external P-input. In a medium loaded ditch the internal P-concentration did not change significantly. In a high loaded ditch increasing internal P-concentrations over time were observed, towards P-saturation of the benthic community.     

The Effects of l-Glutamate and trans-(±)-1-Amino-1,3-Cyclopentanedicarboxylate on Phosphoinositide Hydrolysis Can Be Pharmacologically Differentiated

Abstract: The excitatory amino acid analogues l -glutamate ( l -Glu), l -aspartate ( l -Asp), d -Asp, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylate ( trans -ACPD) stimulate the hydrolysis of phosphoinositides (PI). In the present studies, the effects of noncompetitive and competitive inhibitors on PI hydrolysis stimulated by excitatory amino acid analogues were examined. When agonist and inhibitor were added simultaneously to hippocampal tissue, the noncompetitive inhibitor l -2-amino-3-phosphonopropionate ( l -AP3) did not block the effects of l -Glu, l -Asp, or d -Asp at concentrations that block the effects of trans -ACPD by more than 80%. When tissue was pre-incubated with l -AP3, the effects of l -Glu, l -Asp, or d -Asp were blocked (IC₅₀ values between 65 and 210 µ M ). Unlike l -AP3, l -aspartate-β-hydroxamate ( l -AβHA) inhibited PI hydrolysis stimulated by trans -ACPD, l -Glu, l -Asp, or d -Asp when agonist and inhibitor were added simultaneously in hippocampus; its effects were not time-dependent. In cerebellum, both l -AP3 and l -AβHA had agonist activity. Inhibition by the recently identified competitive inhibitor (+)-α-methyl-4-carboxyphenylglycine [(+)-MCPG] of PI hydrolysis was also examined. (+)-MCPG blocked PI hydrolysis stimulated by trans -ACPD, l -Asp, or d -Asp in both hippocampus and cerebellum (IC₅₀ values between 220 and 1,700 µ M ). The effects of (+)-MCPG were consistent with a competitive mechanism of action. (+)-MCPG (up to 3 m M ) blocked PI hydrolysis stimulated by l -Glu by less than 25% in both hippocampus and cerebellum.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.