Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the beta-adrenergic agonist, terbutaline and 12-O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC50 values for substance P inhibition of [3H]PC release were 10 microM for basal release, 40 microM for TPA-induced release and 50 microM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu5, MePhe8, MeGly9]substance P [5-11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.     

Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a 67-kDa S6 protein kinase from rat liver.

Fractionation of rat liver cytosol on DEAE-cellulose resolved two S6 kinases eluting at 25 mM KCl (peak I) and 100 mM KCl (peak II). The apparent molecular weights of the peak I and peak II kinases are 26,300 and 67,000, respectively. The peak II kinase was further purified and characterized. Incubation of the kinase with [gamma-32P] ATP and Mg2+ resulted in the incorporation of 32P predominantly into a 67-kDa band. Optimal activity of the kinase was observed in the presence of 5 mM Mg2+ and in the pH range of 8.0-8.5. The Km for ATP and 40S subunit were 7.3 microM and 1.5 microM, respectively. The Mg(2+)-stimulated kinase activity was inhibited by various divalent metals, NaF, and polyamines. The properties of the peak II S6 kinase are very similar or identical to the previously described mitogen-activated S6 protein kinase and may represent the nonactivated form of this enzyme.     

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.     

Inhibitory effects of 9-beta-D-xylofuranosyladenine 5'-triphosphate on DNA-dependent RNA polymerase I and II from cherry salmon (Oncorhynchus masou)

In order to determine the mode of action of cytostatic 9-beta-D-xylofuranosyladenine (xylo-A), the inhibitory effects of 9-beta-D-xylofuranosyladenine 5'-triphosphate (xylo-ATP) on DNA-dependent RNA polymerases I and II purified from cherry salmon (Oncorhynchus masou) liver nuclei were studied. This nucleotide showed strong inhibitory action on both RNA polymerases I and II. The K1 values are 14 microM for polymerase I and 5 microM for polymerase II (Km values of ATP are 37 microM for polymerase I and 40 microM for polymerase II). The mode of xylo-ATP was competitive with respect to the incorporation of AMP into RNA and non-competitive to UTP and CTP.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).     

Kinetics of rat liver GM2 ganglioside-beta-D-N-acetylhexosaminidase

The kinetics of beta-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of beta-D-N-acetylhexosaminidase results in a decrease in specific activity against GM2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 microM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/mumol 4-MU-GlcNAc) and the Km was 5 microM.At substrate concentrations greater than 50 microM, the Vmax was 7 pmol GM2/mumol 4-MU-GlcNAc and the Km was 14 microM. The critical micelle concentration of GM2 ganglioside was 20-25 microM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10-15 microM from electrical conductivity measurements which also showed the end of the monomer-micelle transition to occur at 40-50 microM GM2. The increasing excess of micellar substrate at greater than 50 microM GM2 explains the discontinuity in the kinetic plots. Sodium taurocholate had a critical micelle concentration of 9-11 mM using pinacyanol chloride and 2.5-3 mM using electrical conductivity. When included in the assay mixture at a concentration of 10 mM, sodium taurocholate produced a linear kinetic plot. This is probably due to the formation of mixed micelles of detergent and GM2 ganglioside. The Vmax was 200 pmol GM2/MUmol 4-MU-GlcNAc and the Km was 93 microM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.     

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5'')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.     

Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.     

Interaction between trypsin-like enzyme from Streptomyces erythraeus and chicken ovomucoid

Chicken ovomucoid (CO), an effective inhibitor of bovine trypsin, has a reactive site in each of three tandem domains. When CO was subjected to inhibition assay by the method of Green and Work, the second domain (CO Domain II) inhibited bovine trypsin but not TLE-Se, a trypsin-like enzyme from Streptomyces erythraeus, and the first domain (CO Domain I) inhibited neither bovine trypsin nor TLE-Se. However, when the interaction between CO and TLE-Se was analyzed by means of a Lineweaver-Burk plot, it was found that the ovomucoid exhibited competitive inhibition of the bacterial protease at pH 8.0 (Ki = 5.2 microM). In this case, the reactive-site peptide bonds of the first and second domains were specifically hydrolyzed. The isolated CO Domain I also exhibited competitive inhibition of TLE-Se (Ki = 3.1 microM), which specifically hydrolyzed its reactive-site peptide bond.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Photoaffinity labeling of brain glutamate dehydrogenase isoproteins with an azido-ADP.

The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-diphosphate (8N3ADP). 8N3ADP, without photolysis, mimicked the activatory properties of ADP on GDH I and GDH II activities, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [alpha-32P]8N3ADP occurred at around 40 approximately 50 microM photoprobe with apparent Kd values near 25 and 40 microM for GDH I and GDH II, respectively. Photoinsertion of [alpha-32P]8N3ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affinity chromatography and reversed-phase high performance liquid chromatography, photolabel-containing peptides generated by tryptic digestion were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as in the region containing the sequence, EMSWIADTYASTIGHYDIN. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as ADP. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide with the residues Glu179-Asn197 is within the ADP binding domain of the brain GDH isoproteins.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Comparative specificity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic fluorogenic substrates

Homogeneous porcine calpain (Ca2+-dependent cysteine proteinase) was found to hydrolyze a variety of peptides and synthetic substrates. Leu-Trp-Met-Arg-Phe-Ala, eledoisin-related peptide, alpha-neoendorphin, angiotensin I, luteinizing hormone-releasing hormone, neurotensin, dynorphin, glucagon, and oxidized insulin B chain were cleaved with a general preference for a Tyr, Met, or Arg residue in the P1 position preceded by a Leu or Val residue in the P2 position. No great difference in specificity was found between low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II. 4-Methylcoumaryl-7-amide (MCA) derivatives having a Leu(or Val)-Met(or Tyr)-MCA or a Leu-Lys-MCA sequence were also cleaved by either calpain I or calpain II with preference for Leu over Val by a factor of 9 to 16. Calpains I and II showed similar but not identical kinetic behavior for individual substrates. The Km and kcat values ranged from 0.23 to 7.08 mM and 0.062 to 0.805 s-1 for the calpains, while kcat/Km values for the calpains were only 1/433 to 1/5 of those for papain with a given substrate. With succinyl-Leu-Met(or Tyr)-MCA, calpains I and II were half-maximally activated at 12 and 260 microM Ca2+, respectively, and competitively inhibited by leupeptin (Ki = 0.32 microM for I and 0.43 microM for II) or antipain (Ki = 1.41 microM for I and 1.45 microM for II). Thus, this is the first report describing the specificity and kinetics of calpains I and II.     

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus)

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.     

Soluble and particulate Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase in bovine brain

Ins(1,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 microM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85-90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCl. Two soluble enzymes (type I and type II) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 mumol.min-1 mg protein-1 for type I and were 10-20 times lower for type II. Type I and type II Ins(1,4,5)P3 5-phosphatase displayed different Km values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1,3,4,5)P4; in contrast, type II specifically dephosphorylated Ins(1,4,5)P3 but not Ins(1,3,4,5)P4. Type I Ins(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1,4,5)P3 5-phosphatase that was solubilized by cholate. Km values for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 microM and 1 microM, respectively. Similar values were obtained with particulate Ins(1,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type II phosphatase.     

Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.