Purification and characterization of cytosolic diacylglycerol kinases of human platelets

Three isozymes of diacylglycerol kinase (DGK), DGK-I, DGK-II, and DGK-III, were purified from the cytosol of human platelets by successive chromatography on DEAE-cellulose, Ultrogel AcA34, heparin-Sepharose, ATP-agarose, Mono Q, phenyl-Superose, HCA-hydroxyapatite, Wakopak G40, and TSK-3000SW columns. Two DGK species (DGK-I and DGK-III) were purified to apparent homogeneity, and upon SDS-polyacrylamide gel electrophoresis, they showed a single band of apparent molecular mass of 152 kDa (DGK-I) or 58 kDa (DGK-III). The peptide mapping analysis showed that DGK-I and DGK-III are structurally different. DGK-II was only partially purified, and its apparent Mr was estimated to be 75,000 by gel filtration. The specific enzyme activities of the three isozymes were increased 1,480-fold (DGK-I), 690-fold (DGK-II) and 2,100-fold (DGK-III) over original platelet cytosol. The activities of DGK-II and DGK-III were markedly enhanced by the presence of deoxycholate or phosphatidylserine, whereas DGK-I activity was not much affected by the anionic compounds. All of the three activities were strongly suppressed by phosphatidylcholine. Triton X-100 and octyl glucoside were strongly inhibitory to all of the enzymes, although to different extents. The DGK inhibitor, R59022, inhibited DGK-II and to a lesser extent DGK-III, but little affected DGK-I activity. DGK-I was much more heat-stable than DGK-II and DGK-III. The Km values for ATP were 150 microM for DGK-I, 245 microM for DGK-II, and 450 microM for DGK-III. The apparent Km values for suspended diolein were not much different among the DGKs and were in the range of 50-80 microM. These observations indicate that human platelet cytosol contains DGK isozymes with different enzymological properties. Furthermore, the three DGKs isolated from human platelets were found not to cross-react with the antibody raised against porcine brain 80-kDa DGK, thus indicating that human platelets contain novel species of DGK.     

Isozymic forms of protein kinase C in regenerating rat liver

The expression of multiple forms of protein kinase C (PK-C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK-C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK-C caused by phorbol 12-myristate 13-acetate (PMA) was concentration-dependent and was due to translocation of isozyme III, because type II was insensitive to 5 x 10(-8) M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.     

Characterization and partial purification of rat submandibular gland protein kinase C

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.     

Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.     

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol.

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.     

Irreversible inactivation of diacylglycerol kinase-II requires a mediator.

Cytosolic diacylglycerol kinase was irreversibly inactivated by 5'-AMP since the enzyme remained less active after the removal of 5'-AMP by P-10 gel chromatography. The inactivation was time-dependent, suggesting the involvement of a covalent bond modification. A reconstitution experiment detected a rat brain cytosolic mediator for the effect of 5'-AMP. A protein kinase rich fraction prepared from rat liver was also capable of restoring the sensitivity of diacylglycerol kinase-II to 5'-AMP. We propose that 5'-AMP-activated protein kinase is the mediator which inactivates diacylglycerol kinase-II, possibly by phosphorylation.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.     

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.     

Evidence that cyclic AMP-induced inhibition of phosphatidylcholine biosynthesis is caused by a decrease in cellular diacylglycerol levels in cultured rat hepatocytes.

The mechanism by which glucagon and cAMP analogues inhibit phosphatidylcholine biosynthesis was investigated in rat hepatocytes. The studies were facilitated by preparation of an antibody to a synthetic peptide (D-F-V-A-H-D-D-I-P-Y-S-S-A) corresponding to residues 164-176 of CTP:phosphocholine cytidylyl-transferase. The antibody, which was purified by affinity chromatography, quantitatively immunoprecipitated cytidylyltransferase from rat liver cytosol. Various analogues of cAMP had no effect on the labeling of cytidylyltransferase with 32Pi in rat hepatocytes. Nor did the cAMP analogues have any effect on the distribution of cytidylyltransferase between cytosol and membranes. These results indicate that the supply of CDP-choline does not limit phosphatidylcholine biosynthesis in hepatocytes treated with cAMP analogues. A decreased supply of diacylglycerol was considered as an alternative mechanism for inhibition of phosphatidylcholine biosynthesis. An approximately 30% decrease in diacylglycerol concentration was observed in hepatocytes treated with the cAMP analogues or glucagon, compared with controls. A similar decrease of phosphatidylcholine biosynthesis was observed. The cAMP-mediated decrease in diacylglycerol levels and inhibition of phosphatidylcholine biosynthesis were reversed by addition of 0.5-1.5 mM oleic acid to the treated hepatocytes. A correlation coefficient of 0.93 was calculated between the levels of diacylglycerol and the rate of phosphatidylcholine biosynthesis. In another approach, the diacylglycerol levels were increased by an inhibitor of diacylglycerol lipase (U-57908) which also reversed the cAMP effects on diacylglycerol levels and phosphatidylcholine biosynthesis. We conclude that the cAMP-mediated inhibition of phosphatidylcholine biosynthesis was not due to an effect on the phosphorylation of cytidylyltransferase. Instead, phosphatidylcholine biosynthesis appears to be inhibited due to a decreased level of diacylglycerol, a substrate for CDP-choline: 1,2-diacylglycerol cholinephosphotransferase.     

Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart

Fractionation of cytosolic sphingosine kinase (SKase) activity by gel filtration chromatography gave rise to a 96-kDa peak that contained only the SK2 form of SKase (by Western analysis) and a broad ca. 46 kDa peak that contained only SK1 forms. SK2 appeared to have a bound accessory protein. When tested with the classic SKase inhibitor dimethylsphingosine (DMS), SK1 was extensively inhibited; however, SK2 was not inhibited but unexpectedly was activated. Activation of SK2 was the result of DMS enhancing the affinity of the enzyme for sphingosine, and, at low concentrations of ATP and sphingosine, activated by more than 100%. Activation of SK2 could be demonstrated in the cytosolic fraction indicating it was unrelated to the purification step. The immunomodulator FTY720 also activated SK2 (although to a lesser extent), but was a potent inhibitor of SK1. SK2 from rat liver and spleen was also not inhibited by DMS. L-Sphingosine and to a lesser extent dihydrosphingosine and phytosphingosine were effective inhibitors of both forms.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.

Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.     

Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase)

High performance anion-exchange chromatography was used to separate two carnosine-hydrolysing dipeptidases from hog kidney. Both enzymes (peaks I and II) were cytosolic and were activated and stabilized by Mn2+ and dithiothreitol. Peak I had a narrow specificity when assayed without added metal ions, but a broad specificity in the presence of Mn2+ or Co2+. Peak II was inactive unless both Mn2+ and dithiothreitol were present. Bestatin and leucine inhibited peak II, but not peak I. Peak I had a Km of 0.4 mM carnosine, a pI of 5.5 and a Mr of 57,000. Peak II had a Km of 5 mM carnosine, a pI of 5.0 and a Mr of 70,000. Hog and rat brain and liver carnosinase activity was completely inhibited by bestatin, indicating that these organs contained peak II, with little or no peak I enzyme. Hog kidney peak I contained the classical carnosinase of Hanson and Smith, who first described this enzyme. It also contained activity against homocarnosine ("homocarnosinase") and showed "manganese-independent carnosinase" activity. These three activities could not be separated using 8 different chromatographic procedures; it was concluded that they are attributable to one enzyme. It is recommended that the name carnosinase be retained for this enzyme and the names "homocarnosinase" and "manganese-independent carnosinase" be withdrawn. The properties of hog kidney peak II closely resembled those of human tissue carnosinase (also known as prolinase, a non-specific dipeptidase), mouse "manganese-dependent carnosinase" and a rat brain enzyme termed "beta-Ala-Arg hydrolase". Since these terms appear to represent closely related enzymes with broad specificity, the recommended name for each is "non-specific cytosolic dipeptidase".     

Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain

Three types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by beta I and beta II sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1,2-didecanoylglycerol and 1,2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of protein kinase C by cis- and trans-fatty acids and its potentiation by diacylglycerol

Both cis- and trans-unsaturated but not saturated fatty acids activated protein kinase C purified to apparent homogeneity from rat brain. Fatty-acid-induced enzyme activation was not more than additive with that by phospholipids and was potentiated by diacylglycerol. Recently, we demonstrated that cis- and trans-unsaturated fatty acids induced platelet aggregation and phosphorylation of specific proteins. Both events were potentiated by a cell-permeable diacylglycerol [(1987) Biochem. Biophys. Res. Commun. 149, 762-768]. Thus, trans-unsaturated fatty acids may provide useful experimental tools for the study of protein kinase C activation in vitro and in vivo. Our results suggest that fatty acids and diacylglycerol may synergistically be involved in hormonal stimulation of protein kinase C, as certain hormonal stimuli cause release of diacylglycerol and fatty acids from phospholipids by parallel activation of phospholipases C and A2.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase

Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.