Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.

Experiments were performed to investigated the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca(2+)-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca(2+)-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca(2+)-dependent, 150-kD protein kinase.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation

Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO₂ or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO₂ was removed in continuous light, and was reactivated when CO₂ was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots.     

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.     

A Retinal Calmodulin-Dependent Kinase: Calcium/ Calmodulin-Stimulated and -Inhibited States

Abstract: A calcium/calmodulin-dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50-kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50-kD and 60-kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto-phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin-dependent to a calcium/calmodulin-inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin-independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.     

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.     

高温胁迫下葡萄叶片蛋白激酶的诱导形成与活性变化

以"京秀"葡萄(Vitis vinifera L.cv.Jingxiu)幼苗为试材,研究了高温胁迫激活的蛋白激酶的类型和活性.结果表明,高温胁迫10～60min明显地激活了一个分子量约为52 kD的蛋白激酶,该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白(MBP)磷酸化,在放射自显影中表现出很高的放射活性,而对凝胶中的组蛋白-Ⅲ(histone-Ⅲ)则没有这样的作用.在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性,而对histone-Ⅲ却无作用.Ca2 对其活性变化无显著影响.酪氨酸特异性蛋白磷酸酶(YOP)对该激酶的活性有显著的钝化作用.结果表明该52 kD蛋白激酶是MAPK家族中的一种.     

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

Evidence is accumulating that the activity of spinach (Spinacia oleracea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and in vivo by protein phosphorylation. From the present study we report the partial purification of the two protein factors needed for NR inactivation. We identified NR-protein kinase (NR-PK) as a calcium-dependent and metabolite-regulated protein kinase and have provided additional evidence that phosphorylation of NR is necessary but not sufficient to inactivate the enzyme. The inhibitor protein required for inactivation of phospho-NR was purified 625-fold by polyethylene glycol fractionation and sequential column chromatography. Using partially purified inhibitor protein and NR-PK, we characterized NR inactivation (increased sensitivity to Mg2+ inhibition) in a reconstituted in vitro system. NR-PK activity was inhibited by a variety of metabolic phosphate esters including di-hydroxyacetone phosphate, glucose-6-phosphate, and fructose-1,6-bisphosphate. Light-to-dark transition experiments with a starchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosphate esters during the photoperiod, indicated that NR inactivation in vivo might, indeed, be down-regulated by metabolites. Additionally, we postulate that cytosolic free calcium could play an important role in the regulation of NR activity in vivo.     

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

Annual changes of activity of sucrose-phosphate synthase (SPS) from spruce (Picea abies [L.] Karst.) needles were studied with respect to three regulatory levels: metabolic fine control, covalent modification (phosphorylation), and protein amount. Glucose-6-phosphate served as an allosteric activator of spruce SPS by shifting the Michaelis constant for the substrate fructose-6-phosphate from 4.2 to 0.59 mM, whereas inorganic phosphate competitively inhibited this activation. The affinity for the other substrate, UDP-glucose, was unaffected. Incubation of the crude extract with ATP resulted in a time- and concentration-dependent decrease of the maximal velocity of SPS. This inactivation was sensitive to staurosporine, a potent protein kinase inhibitor, indicating the participation of a protein kinase. Probing SPS protein with heterologous antibodies showed that the subunit of spruce SPS is an approximately 139-kD protein and that changes in the extractable activity during the course of a year were correlated with the amount of SPS protein. High SPS activities in winter were paralleled by increased levels of the activator glucose-6-phosphate and the substrate fructose-6-phosphate, indicating a high capacity for sucrose synthesis that may be necessary to maintain photosynthetic CO2 fixation in cold-hardened spruce needles.     

The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.     

Isolation and study of cAMP-independent chromatin protein kinases from brain cells]

Two cAMP-independent protein kinases were purified from rat brain neuron chromatin by using extraction with ammonium sulfate with subsequent chromatography on DEAE-Sephadex A-25 and Sephadex G-150. These enzymes were identified as casein kinases NI and NII, respectively. The molecular masses of the proteins as determined by gel filtration are 4500 and 130 Da. Casein kinase NII utilizes ATP (Km = 7.5 mM) and GTP (Km = 8.5 mM) as substrates, while casein kinase NI utilizes only ATP (Km = 6 mM). The activities of the both enzymes are inhibited by Mn2+ and Ca2+, while heparin (1 microgram/ml) inhibits only casein kinase NII. The memory stimulator ethymizol (ethylnorantipheine) increases the activity of casein kinase NII only when brain proteins extracted by 0.35 M NaCl or rat liver HMG-proteins are used as reaction substrates. This substance has no effect on the phosphorylation of casein and histone HI. The role of casein kinase NII of neuronal chromatin in the realization of stimulatory effects of physiologically active substances on RNA synthesis is discussed.     

In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase

(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.     

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.     

IAA对小麦胚芽鞘质膜蛋白磷酸化的影响

磷酸化／ 脱磷酸化机制是众多信号转导过程中的重要环节,很多信号物质被细胞受体识别后引发蛋白激酶和蛋白磷酸酶活性变化,通过磷酸化／ 脱磷酸化进一步调节多种酶活性而产生各种生理效应。在对生长素ＩＡＡ 的信号转导的研究中,发现ＩＡＡ 处理的小麦胚芽鞘质膜蛋白中蛋白激酶的活性和蛋白磷酸化程度都发生改变,并找到两种受到调节的蛋白激酶。钙离子通道抑制剂ＬａＣｌ３ 阻断了ＩＡＡ 的这种作用,表明Ｃａ２＋参与了ＩＡＡ的信号转导过程。     

Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity

The neuroprotective effects of lithium, a mood stabilizer, against glutamate-induced excitotoxicity in rat cortical neurons were associated with a decrease in Tyr1472 phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit and a loss of receptor activity. Since this receptor tyrosine phosphorylation is mediated by the Src-family tyrosine kinases, we investigated the effects of lithium on the Src kinase activity. Levels of phosphorylated Src kinase at Tyr416, an index of Src activation, were reduced after treatment with LiCl (1 mM) for more than 3 days. Protein levels of Src-family kinases such as Src, Fyn, and Yes were unchanged by lithium treatment. The activities of cytosolic protein tyrosine kinase and protein phosphatase were also unchanged by lithium treatment, indicating the selectivity and the modulation. Moreover, the levels of postsynaptic densities (PSD) and SynGAP, the scaffolding proteins of the NMDA receptor complex, were unaltered by lithium. A Src kinase inhibitor, SU6656, and an NR2B antagonist, ifenprodil, partially blocked glutamate excitotoxicity. Our results suggest that lithium-induced inactivation of Src kinase contributes to this drug-induced NMDA receptor inhibition and neuroprotection against excitotoxicity.     

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.     

Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.