Zinc and calcium ions cooperatively modulate ADAMTS13 activity

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

Characterization of an endogenous inhibitor of lung 15-hydroxyprostaglandin dehydrogenase

An endogenous inhibitor of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was isolated from the 105,000 X g supernatant fraction of lungs of pregnant rabbits following DEAE chromatography. The material was heat stable and was resistant to pronase treatment. The inhibitor contained a mixture of saturated and mono-unsaturated fatty acids and cholesterol with palmitate and oleate representing the major fatty acids in the inhibitory factor. The factor inhibited prostaglandin dehydrogenase activity but had only minor effects on the activity of NAD+-dependent alcohol and lactate dehydrogenases or the NADP+-dependent isocitrate dehydrogenase. In an attempt to develop a greater understanding of the inhibitory action of fatty acids on prostaglandin dehydrogenase activity, a variety of standard fatty acids were examined for their ability to decrease enzymic activity. Oleate and palmitate inhibited enzymic activity by 70% at 10 microM, whereas arachidonate and myristate were only 30% inhibitory at this concentration. A comparison among the 18-carbon-containing fatty acids demonstrated that oleate was more potent than linoleate and linolenate in inhibiting prostaglandin dehydrogenase activity. The coenzyme A derivatives of oleate, linoleate and linolenate were less inhibitory than the free fatty acids.     

Characterization of the heme environment in Arabidopsis thaliana fatty acid alpha-dioxygenase-1

Plant alpha-dioxygenases (PADOX) are hemoproteins in the myeloperoxidase family. We have used a variety of spectroscopic, mutagenic, and kinetic approaches to characterize the heme environment in Arabidopsis thaliana PADOX-1. Recombinant PADOX-1 purified to homogeneity contained 1 mol of heme bound tightly but noncovalently per protein monomer. Electronic absorbance, electron paramagnetic resonance, and magnetic circular dichroism spectra showed a high spin ferric heme that could be reduced to the ferrous state by dithionite. Cyanide bound relatively weakly in the ferric PADOX-1 heme vicinity (K(d) approximately 10 mm) but did not shift the heme to the low spin state. Cyanide was a very strong inhibitor of the fatty acid oxygenase activity (K(i) approximately 5 microm) and increased the K(m) value for oxygen but not that for fatty acid. Spectroscopic analyses indicated that carbon monoxide, azide, imidazole, and a variety of substituted imidazoles did not bind appreciably in the ferric PADOX-1 heme vicinity. Substitution of His-163 and His-389 with cysteine, glutamine, tyrosine, or methionine resulted in variable degrees of perturbation of the heme absorbance spectrum and oxygenase activity, consistent with His-389 serving as the proximal heme ligand and indicating that the heme has a functional role in catalysis. Overall, A. thaliana PADOX-1 resembles a b-type cytochrome, although with much more restricted access to the distal face of the heme than seen in most other myeloperoxidase family members, explaining the previously puzzling lack of peroxidase activity in the plant protein. PADOX-1 is unusual in that it has a high affinity, inhibitory cyanide-binding site distinct from the distal heme face and the fatty acid site.     

Charge effects on phospholipid monolayers in relation to cell motility

A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 microM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and stearate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59 +/- 3%, S.E.) and kidney (76 +/- 3%), with considerably lower overall activity in kidney (57-375 nmol/h per g of tissue) than in liver (394-1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 micrograms/dl, and of liver vitamin A concentrations from 0 to 32 micrograms/g. Pig liver retinyl ester hydrolase differs from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability.     

Tropomodulin contains two actin filament pointed end-capping domains

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.     

Composition of cholesterol esters in blood plasma of mouse, rat and rabbit

Mixtures of cholesterol estes were extracted from the blood plasma of mouse (C3H/He, C57BL/6J and Jcl : ICR), rat (Wistar) and rabbit (JW-NIBS/Rabiton) and purified by thin-layer chromatography. Fatty acid moieties of the esters were methylated with BF3-methanol and analyzed quantitatively by gas chromatography. Although linoleate was most abundant (over 30% of the total esters) and myristate was found only in traces, the composition of the esters varied markedly among the species. Mice had 15.3 to 17.4% of arachidonate, but only a trace of linolenate and no stearate. In rats, arachidonate and linoleate were found in almost equal quantities, but stearate, linolenate and docosahexaenoate were present in only trace amounts. Rabbits had more palmitate and oleate and less arachidonate and eicosapentaenoate than the other two animals, but no docosahexaenoate.     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Evidence that bacterial cyanide oxygenase is a pterin-dependent hydroxylase

The soluble cell-free fraction (150,000g high-speed supernatants [HSS]) of Pseudomonas fluorescens NCIMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O(2) or NADH uptake, or by conversion of radioactive K(14)CN to (14)CO(2), was detected at micromolar concentrations (apparent half-saturation constant, 4 microM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (<500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

The chemical synthesis of ecdysone 22-long-chain fatty acyl esters in high yield

Eight ecdysone 22-long-chain fatty-acyl esters (laurate, myristate, palmitate, stearate, oleate, linoleate, linolenate and arachidate) have been chemically synthesised in high yield. Ecdysone was first converted to the 2,3-acetonide derivative and then acylated selectively at C-22 with the appropriate acyl anhydride. The protecting acetonide group is then removed by mild acid treatment to yield the ecdysone 22-acyl ester. Reaction conditions have been optimised to maximise the overall yield (ca. 70%). The ecdysone acyl esters and their 2,3-acetonide derivatives have been characterised by 1H- and 13C-NMR and high-resolution FAB-mass-spectrometry.     

The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.     

Phosphorylase kinase phosphorylation of skeletal-muscle troponin T.

When [14C]diacylgalactosylglycerol was added to isolated pea or lettuce chloroplasts linolenate synthesis was seen. The desaturation of [14C]linoleate in diacylgalactosylglycerol to [14C]linolenate was stimulated by the addition of a soluble protein fraction containing lipid-exchange activity. Other [14C]acyl lipids were ineffective, except that [14C]phosphatidylcholine in the presence of UDP-galactose and sn-glycerol 3-phosphate could also supply [14C]linoleate for desaturation. These results are consistent with a role of diacylgalactosylglycerol in linolenate synthesis, as indirectly suggested by labelling experiments.     

Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1.

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.     

Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.     

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).