Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.     

Participation of the tryptophan synthase inactivating system from yeast in the activation of chitin synthase

The previously described tryptophan synthase “inactivase II”, a proteolytic enzyme from yeast, exhibits high activity in the activation of chitin synthase. Tryptophan synthase inactivase I shows essentially no activity.The purified, heat-stable inhibitor of the tryptophan synthase inactivating enzymes also inhibits the activation of chitin synthase. We take these results to mean that the proteolytic inactivation of tryptophan synthase and the proteolytic activation of chitin synthase are catalyzed and regulated by the same protease/inhibitor system     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays in human myometrium during the last trimester of pregnancy (n = 23) and in non-pregnant controls (n = 8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p less than 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p less than 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells. Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p less than 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI2 production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.     

Kinetic studies on inhibition of aminopropyltransferases by aurintricarboxylic acid in vitro

Activities of aminopropyltransferases (spermidine synthase and spermine synthase) were inhibited by aurintricarboxylic acid (ATA). Spermidine synthase was slightly more sensitive to the inhibitor than spermine synthase. These inhibitions were not prevented by 0.15 M NaCl. Inhibition by ATA of spermidine synthase was ‘uncompetitive’ with respect to putrescine and that of spermine synthase was ‘non-competitive’ with respect to spermidine. When the amount of spermidine synthase or spermine synthase was varied, inhibition ratio hardly changed on either case implying no appreciable interaction between ATA and these enzymes.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays inhuman myometrium during the last trimester of pregnancy (n=23) and in non-pregnant controls (n=8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p < 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p < 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells.Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p < 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI₂ production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.     

Glycogen synthase R in livers of starved rats and starved rats given glucose

We reported that when synthase D was converted to synthase I in a rat liver extract, it progressed through a synthase form with activity characteristics which could not be explained by a mixture of synthase D and synthase I (Tan, A. W. H. (1981) Biochem. J. 200, 169-172). In this study we will borrow the "R" nomenclature to describe this "non-D" and "non-I" activity. Using activities measured at five different conditions and simultaneous equations, the amount of the three synthase forms in liver extracts can be estimated. During incubation of the liver extract, the amount of synthase R was found to increase with time and then to decrease as synthase I was generated, a profile typical of an enzyme intermediate. We investigated for the presence of synthase R in rat liver under different in vivo conditions. In contrast to the liver of fed rats which had very little synthase R, the liver of fasted rats was found to have 30% of its synthase in the R form. This synthase R was increased 2-fold when glucose was given and decreased to a very low level when glucagon was given. Synthase I was not detected, even in the livers of starved rats given glucose. Using conditions which were closer to those of the cell, synthase R was found to have relatively high activity, up to 70% that of synthase I. Based on these results, synthase R is proposed to be an active enzyme form responsible for glycogen synthesis in rat liver.     

Not prostacyclin synthase but prostaglandin endoperoxide synthase increases with human placental development

Prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantitated with specific immunoradiometric assays in microsomes from human placentae (n = 20) obtained from 7 up to 17 weeks of gestation. Over that period, wherein trophoblastic invasion of the uterine spiral arteries occurs, the placentae showed a significant increase in concentrations of PGH synthase (r = 0.73, p less than 0.001; n = 20), but not in those of PGI synthase. While the variation between individual placentae was much larger for PGI synthase than for PGH synthase concentrations, there was no evidence for a large excess of PGI synthase over that of PGH synthase in any of these early placentae. The data indicate, first, that the developing placenta contains PGI synthase, but in amounts which are relatively small and do not appear to increase with advancing gestation. Second, they seem to indicate that the capacity for bioconversion of arachidonic acid into prostaglandin endoperoxides increases markedly with placental development.     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.     

Prostaglandin H synthase: an example of enzymic symbiosis

Reaction conditions which promote the heme-dependent peroxidase activity of prostaglandin H synthase appear to stimulate the heme-dependent cyclooxygenase activity also present in the synthase, even though the cyclooxygenase requires hydroperoxide for activity. However, aspirin-treated synthase, which retains only peroxidase activity, inhibited the cyclooxygenase activity of untreated synthase in the manner observed with similar levels of glutathione peroxidase. Any stimulatory effect of the synthase peroxidase on the synthase cyclooxygenase is thus likely to involve an intramolecular mechanism. Participation of peroxidase intermediates (Compounds I and II) in the initiation of a cyclooxygenase free radical chain reaction may provide an intramolecular mechanism for stimulation of the synthase cyclooxygenase by the synthase peroxidase.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

Glycogen synthase phosphatase of rat liver. Its separation from phosphorylase phosphatase on DE-52 columns.

Glycogen synthase D was prepared from rat liver by chromatographing the glycogen pellet on DE-52 columns. It was free of glycogen and phosphorylase and converted readily into synthase I upon incubation with glycogen synthase phosphatase. With this synthase D as substrate, the identity of rat liver glycogen synthase phosphatase was studied by means of DE-52 column chromatography. Under the conditions developed, synthase phosphatase emerged from the columns as a sharp, single peak, and phosphorylase phosphatase came off later. The two phosphatases were also different from each other in stability, synthase phosphatase being less stable than phosphorylase phosphatase.     

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.     

Regulation of glycogen synthase activity in muscle by a C-terminal part sequence of human growth hormone

The synthetic peptide hGH 177–191, corresponding to the last 15 residues at the carboxyl terminus of human pituitary growth hormone, promotes the conversion of glycogen synthase α to glycogen synthase b in muscle. When injected, the peptide was found to produce inactivation of glycogen synthase phosphatase activity in rat skeletal muscle. The time course of phosphatase inactivation was closely correlated with that for glycogen synthase. The peptide had no effect either on muscle 3′,5′-cyclic AMP levels or on synthase kinase activity. These results can be explained in terms of a dynamic cycle of interconversion of synthase between active and inactive forms, by the simultaneous action of synthase kinases and synthase phosphatases. A decrease in the ratio of phosphatase to kinase activity would result in a decrease in the steady-state level of synthase α activity.     

Aquifex aeolicus 3-Deoxy-d-manno-2-Octulosonic Acid 8-Phosphate Synthase: A New Class of KDO 8-P Synthase?

The relationship between 3-deoxy-D-manno-2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis. Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E. coli enzyme. Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor.     

Purification and characterization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG.

Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.     

Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach

Cysteine synthase (O-acetylserine (thiol) lyase, EC 4.2.99.8) and -cyanoalanine synthase (EC 4.4.1.9) have been isolated from leaves of Spinacea oleracea L. and separated by anion-exchange chromatography. Further separation of one minor and two major isoforms of cysteine synthase was achieved by hydrophobic interaction chromatography and high resolution native electrophoresis (PAGE). Analysis of root material indicated that amongst the multiple isoforms present, a single isoform predominated. Subcellular fractionation studies indicated that one of the major leaf forms, cysteine synthase B, was located in the chloroplast and the other, cysteine synthase B, occurred in the cytoplasm. No specific isoform of cysteine synthase was resolved in the mitochondria, while cyanoalanine synthase was predominantly located in the mitochondrial fraction. Sulphur deprivation decreased cysteine synthase activity, but not cyanalanine synthase activity in both young and mature leaves, although cysteine synthase activity in the roots increased slightly. A selective decrease in cystein synthase B (chloroplastic abundance was observed in mature leaves. Patterns of expression of cysteine synthase in response to S-availability are discussed in relation to possible roles for this enzyme in controlling S-flux through the S-assimilatory pathway.Key words: Cysteine synthase isoforms (expression of), Spinacea oleracea L., sulphur deficiency.      

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with ³²P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [γ-³²P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of M_r 85,000 and 80,000 were present in varying proportions. The ³²P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the homogeneous synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.