Cloning and functional characterization of NtCPK4, a new tobacco calcium-dependent protein kinase

A cDNA clone, encoding calcium (Ca2+)-dependent protein kinase (CDPK or CPK), was isolated from tobacco (Nicotiana tabacum). The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues. Sequence alignment indicated that NtCPK4 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs). Biochemical analyses showed that NtCPK4 phosphorylated itself and calf thymus histones fraction III-S (histone III-S) in a calcium-dependent manner, and the K0.5 of calcium activation was 0.29 microM or 0.25 microM with histone III-S or syntide-2 as substrates, respectively. The Vmax and Km were 588 nmol min-1 mg-1 and 176 microg ml-1, respectively, when histone III-S was used as substrate, while they were 2415 nmol min-1 mg-1 and 58 microM, respectively, with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue, as shown by capillary electrophoresis analyses. All of these data demonstrated that NtCPK4 was a serine/threonine protein kinase. NtCPK4 as a low copy gene was expressed in all tested organs including the root, leaf, stem, and flower of tobacco, while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli.     

Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco

A cDNA encoding a calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca2+-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as a Ca2+/CaMK since NtCaMK1 phosphorylated itself and histone IIIs as substrate only in the presence of Ca2+/CaM with a Km of 44.5 microm and a Vmax of 416.2 nm min(-1) mg(-1). Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1 microm and a Vmax of 644.1 nm min(-1) mg(-1) when assayed in the presence of Ca2+/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca2+/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (Kd = 5 nm). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.     

3 alpha-hydroxysteroid dehydrogenase activity catalyzed by purified pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

In earlier studies, two distinct molecules, 20 alpha-HSD-I and 20 alpha-HSD-II, responsible for 20 alpha-HSD activity of pig adrenal cytosol were purified to homogeneity and characterized [S. Nakajin et al., J. Steroid Biochem. 33 (1989) 1181-1189]. We report here that the purified 20 alpha-HSD-I, which mainly catalyzes the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, catalyzes 3 alpha-hydroxysteroid oxidoreductase activity for 5 alpha (or 5 beta)-androstanes (C19), 5 alpha (or 5 beta)-pregnanes (C21) in the presence of NADPH as the preferred cofactor. The purified enzyme has a preference for the 5 alpha (or 5 beta)-androstane substrates rather than 5 alpha (or 5 beta)-pregnane substrates, and the 5 beta-isomers rather than 5 alpha-isomers, respectively. Kinetic constants in the reduction for 5 alpha-androstanedione (Km; 3.3 microM, Vmax; 69.7 nmol/min/mg) and 5 beta-androstanedione (Km; 7.7 microM, Vmax; 135.7 nmol/min/mg) were demonstrated for comparison with those for 17 alpha-hydroxyprogesterone (Km; 26.2 microM, Vmax; 1.3 nmol/min/mg) which is a substrate for 20 alpha-HSD activity. Regarding oxidation, the apparent Km and Vmax values for 3 alpha-hydroxy-5 alpha-androstan-17-one were 1.7 microM and 43.2 nmol/min/mg, and 1.2 microM and 32.1 nmol/min/mg for 3 alpha-hydroxy-5 beta-androstan-17-one, respectively. 20 alpha-HSD activity in the reduction of 17 alpha-hydroxyprogesterone catalyzed by the purified enzyme was inhibited competitively by addition of 5 alpha-DHT with a Ki value of 2.0 microM. Furthermore, 17 alpha-hydroxyprogesterone inhibited competitively 3 alpha-HSD activity with a Ki value of 150 microM.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.     

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Biochemical characterization of a calcium-sensitive protein kinase LeCPK2 from tomato

LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca(2+)-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The K(m) value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 microg/ml and 89.52 microM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.     

Synthesis and transport characteristics of photoaffinity probes for the hepatocyte bile acid transport system

In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes.     

Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase

The kinetic mechanism of guinea pig neutrophil 5-lipoxygenase was investigated using a continuous spectrophotometric assay that monitors product diene formation at 236 nm due to substrate oxygenation. Progress curves for reactions with both arachidonic acid and eicosapentaenoic acid are characterized by 1-3-min lag phases in the attainment of steady-state velocities and product inhibition, as indicated by the total cessation of the reaction prior to complete depletion of substrate. The dependence of the steady-state velocity on arachidonic acid concentration appears to follow Michaelis-Menten kinetics, with Vmax = 4.2 +/- 0.4 nmol of 5-hydroxy-6,8,11,14-eicosatetraenoic acid/min/mg of protein and Ks = 25 +/- 4 microM. The addition of Ca2+ results in an overall activation: lag phases are shortened to 10-20 s, Vmax increases to 24 +/- 2 nmol/min/mg of protein, and Ks decreases to 7.7 +/- 1.7 microM; and a change in a mechanism to one involving substrate inhibition (Kss = 13 +/- 1 microM). The observed activation by Ca2+ has a half-maximal response at around 30 microM. In the presence of Ca2+, ATP causes an increase in Vmax to 30 +/- 4 nmol/min/mg of protein without changing Ks or Kss and a reduction of the lag to less than 5 s. The half-maximal response for ATP is 31 +/- 7 microM. Oxygenation of eicosapentaenoic acid in the presence of Ca2+ and ATP occurs with similar kinetics, except for significantly less substrate inhibition: Vmax = 31 +/- 6 nmol/min/mg of protein, Ks = 7 +/- 1 microM, and Kss = 33 +/- 2 microM. This is the first report suggesting a kinetic mechanism for 5-lipoxygenase, which accounts for substrate inhibition, regulation by Ca2+, and ATP and substrate specificity.     

Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.     

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.     

Degradation of kyotorphin by a purified membrane-bound-aminopeptidase from monkey brain: potentiation of kyotorphin-induced analgesia by a highly effective inhibitor, bestatin

Kyotorphin (Tyr-Arg) was rapidly degraded in rat brain homogenates and the Vmax and Km were 29.4 nmol/mg protein/min and 16.6 microM, respectively. This degradation was effectively inhibited by bestatin (IC 50; 0.08 microM) and p-chloromercuribenzoate (IC 50; 0.70 microM). Kyotorphin was also degraded by a membrane-bound aminopeptidase from monkey brains. The Vmax and Km of kyotorphin degradation by the aminopeptidase were 20.0 nmol/mg protein/min and 29.2 microM, respectively. The degradation of kyotorphin was also inhibited effectively by bestatin (KI; 0.4 microM). Co-administration with bestatin 50 micrograms (i.cist.) potentiated the analgesic effects of kyotorphin (i.cist.) by 4.8 times, and these effects were abolished by pretreatment with naloxone 0.5 mg/kg s.c. These results suggest that potentiation of analgesia by bestatin may be due to the protection against the degradation of kyotorphin and released enkephalin by a membrane-bound aminopeptidase.     

Purification and characterization of the beta-adrenergic receptor kinase

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.