Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L

A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.     

cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli

A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced. The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D. discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM). The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid).     

Elevated Ca2+(i) transients induced by trimethyltin chloride in HeLa cells: types and levels of response

Humans are exposed to organotins, like trimethyltin (TMT) chloride via air, water and food, and intoxication might result in severe health complications. Toxic effects of organotin compounds are well documented, but possible mechanisms remain unclear and only little information is available how organometallic species interact with calcium controlling mechanisms. Therefore, the aim of this work was to investigate the effects of TMT on calcium homeostasis in HeLa S3 cells. Dynamic changes of cytosolic calcium (Ca2+(i)) were monitored using laser-scanning microscopy and fluo-4 loaded cells. Application of TMT resulted in sustained as well as in transient elevations of Ca2+(i). The number of reacting cells was directly correlated to the concentration of TMT used: with 500 microM TMT all cells reacted, with 50 microM TMT 80% and with 5 microM 74%. The fast Ca2+(i)-transients (spikes), measured in single cells, occurred even with 0.25 microM TMT and varied in size and duration. The sustained increase of Ca2+(i), measured as the average over all cells, was dose dependent with an approximately 8% increase for 5 microM TMT, approximately 12.3% for 50 microM and approximately 145% for 500 microM TMT. Moreover, this effect was partly reversible. A second application resulted in a similar sustained rise of Ca2+(i) compared to the first application of TMT, there was also no difference when no calcium was added to the external solution (151+/-10% compared to 145+/-15%; 500 microM TMT). This rise of Ca2+(i) was highly reduced (<10% increase) when the internal calcium stores were depleted before TMT (500 microM) was applied. Our data suggest that TMT influences Ca2+(i)-homeostasis of HeLa S3 cells, which might be related to its toxicity in this cell line.     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

Demonstration and Biochemical Characterisation of Rat Brain NADPH-Dependent Diaphorase

An enzyme responsible for the NADPH-dependent reduction of nitroblue tetrazolium HCl (NBT) has been isolated from rat brain. Although other tetrazolium salts could be utilised, NBT was the preferred substrate, and the enzyme had an absolute requirement for NADPH. An in vitro assay was developed and used to determine the kinetic constants: Km NBT = 17.3 microM; Km NADPH = 1.9 microM, Vmax = 30.8 mumol product produced/min/mg protein. Substrate inhibition by NADPH was observed in some instances. Brain subcellular fractionation indicated highest enzyme activities in the microsomal fraction. Activity was present in all brain regions and in a variety of peripheral tissues. Relative molecular mass determinations of the native enzyme yielded an Mr = 170-180,000. It seems likely that the enzyme activity described in this study relates directly to the histochemical demonstration of brain NADPH-diaphorase-positive neurons. As yet, the natural substrate for the enzyme is unknown. However, the isolation and purification of NADPH-dependent diaphorase may be anticipated to assist in the elucidation of its function in the brain, and in the special characteristics of those neurons that contain the enzyme in abundance.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134.

Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity. The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE. Mn2+ ions as well as thiol groups are required for activity. A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound. Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate. The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.     

A specific enzyme assay for aminopeptidase M in rat brain.

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin > MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6 microM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 microM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 microM) had only a slight effect on the Km of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The Km of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the Km of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.     

Kinetics of gossypol inhibition of bovine lactate dehydrogenase X

Gossypol, a polyphenolic binaphthalene dialdehyde isolated from cotton meal is a potent inhibitor of lactate dehydrogenase-X purified from bovine testis. For the conversion of pyruvate to lactate the IC50 for gossypol is 200 microM for the reverse reaction the IC50 is 12 microM. Gossypol is a competitive inhibitor of NADH, Ki = 30 microM (Km = 17 microM), and NAD+, Ki = 6 microM (Km = 130 microM), and noncompetitive for pyruvate, Ki = 220 microM (Km = 224 microM), and lactate, Ki = 52 microM (Km = 5.6 mM).     

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

Purification and properties of Oxidized and reduced forms of Hepatic fructose 1,6-bisphosphatase

D-Fructose 1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) was isolated from rat liver in two forms: "A," isolated in the presence, and "B," isolated in the absence of dithiothreitol. Both forms had an apparently identical molecular weight of approximately 37,000/subunit and the same Km for fructose 1,6-bisphosphate of 2 microM. However, the Ki of the AMP inhibition of form A was 140 microM and of form B, 370 microM. With form B the same inhibition as with form A was reached by incubating the enzyme with dithiothreitol. The two forms of the enzyme differed in their total, as well as in their number of fast reacting thiol groups. Form A was the more reduced form, exhibiting 22.4 thiol groups/molecule, 2.5 of them fast reacting with 5,5'-dithiobis-(2-nitrobenzoic acid). Only 0.5 fast reacting groups and a total of 19.2 were found with form B. The fast reacting thiol groups disappeared when assayed in the presence of AMP. It is suggested that a redox reaction alters a site that influences the inhibitory action of AMP, so as to regulate the activity of fructose 1,6-bisphosphatase.     

Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides

Purified rat brain cathepsin B (EC 3.4.22.1) converted prodynorphins or proenkephalins to shorter active forms by the preferential removal of C-terminal dipeptides. The substrate affinities for Met-enkephalin-Arg-Phe or -Arg-Gly-Leu were Km 46 and 117 microM, and kcat/Km ratios were 67 and 115 microM-1, min-1, respectively. Met-Enkephalin was inactivated by the same mechanism (Km-450 microM; kcat/Km = 0.12 microM-1 min-1). The comparison of cathepsin B hydrolysis for pro-opioids, a synthetic hexapeptide and its fragments, C-blocked peptides (pro-opioid amides, Met-enkephalin amide, substance P), and bovine myelin basic protein, provided information on the influence of the C-terminal residues on dipeptide release, the rates as correlated to peptide length, and the optimal arrangement of residues favoring scission at the P1-P'1 sites. The brain enzyme was stereospecific and did not act on peptides with C-terminal D-amino acid substituents. Arg hindered and Pro blocked the release of C-terminal dipeptides when in the P'2 positions. The suppression of dipeptide release by agents inhibiting endopeptidase actions such as E-64 and leupeptin, and the endogenous brain factor (cerebrocystatin) point to similar catalytic mechanisms for the exopeptidase action.     

Uptake of gamma-aminobutyric acid by catfish brain

Using homogenates of catfish whole-brain in an isotonic medium, we observed an accumulation of [3H]GABA that was temperature-sensitive and was dependent on the presence of sodium ions, the optimum concentration of which was 75 mM. A kinetic analysis showed that the [3H]GABA uptake mechanism became saturated with increasing GABA concentrations. A high-affinity system, only, was evident whose Km was calculated as 12 microM. Four structural analogues of GABA were found to be competitive inhibitors of uptake, and Ki values were determined. Nipecotic acid (Ki = 1.8 microM) and guvacine (Ki = 3.9 microM) were the most potent compounds, however 2,4-diaminobutyric acid (Ki = 8.9 microM) and beta-alanine (Ki = 55 microM) also had an effect. The characteristics of the uptake mechanism in catfish brain that we have studied are similar to those reported for uptake by mammalian brain except that in the latter, both a high- and a low-affinity transport processes are present. Our data, taken together with what is already known, strongly suggest that the biochemistry of the GABA system in lower vertebrates does not differ significantly from that in mammals.     

Enkephalin analogs as substrates for the assay of brain cysteine proteinase (Cathepsin L) and its endogenous inhibitors

A series of enkephalin-like peptides (X-Tyr-Gly-Gly-R-Pro) were synthesized for assay of cathepsin L and papain. Enzymes acted only at the Gly-Gly bond to release N-terminal dipeptides. When X = dansyl and R = Phe(NO2) the substrate was suited for continuous fluorimetric assay of rat brain cathepsin L (Km 45 microM, kcat/Km 1333 mM-1 sec-1). The substituted pentapeptides provided information on the influence of P2, P2' residues on rates of Gly-Gly cleavage. The synthetic substrate provided rapid and sensitive assays for the brain cathepsin L and its interaction with 13-14 kDa (cerebrocystatin) and 70 kDa (T-kininogen) rat brain inhibitors. The suppression of cathepsin L- or papain-mediated hydrolysis of substrates by inhibitors may be the result of competition between their binding domains at the enzyme catalytic center.     

The role of inositol 1,4,5-trisphosphate 5-phosphatase in inositol signaling in the CNS of larval Manduca sexta

Production of inositol 1,4,5-trisphosphate (IP3) in cells results in the mobilization of intracellular calcium. Therefore, the dynamics of IP3 metabolism is important for calcium dependent processes in cells. This report investigates the coupling of mAChRs to the inositol lipid pathway in the CNS of the larval Manduca sexta. Stimulation of intact abdominal ganglia prelabeled with [3H]-inositol using a muscarinic agonist, oxotremorine-M (oxo-M), increased total inositol phosphate levels in a dose dependent manner (EC50 = 4.23 microM). These inositol phosphates consisted primarily of inositol 1,4-bisphosphate (IP2) and inositol monophosphate (IP1). Similarly, when nerve cord homogenates were provided with [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]-PIP2) (10-13 microM) the predominant products were IP2 and IP1. In contrast, incubation of purified membranes with 1 mM oxo-M in the presence of 100 microM GTP gamma S and [3H]-PIP2 increased IP3 levels, suggesting that the direct activation of phospholipase C (PLC) by mAChRs occurs in a membrane delimited process. Together, these results suggest that in the intact nerve cord and in crude homogenates, a cytosolic 5-phosphatase quickly metabolizes IP3 to produce to IP2 and IP1. This enzyme was kinetically characterized using IP3 (Km = 43.7 microM, Vmax = 864 pmoles/min/mg) and IP4 (Km = 0.93 microM; Vmax = 300pmoles/min/mg) as substrates. The enzyme activity can be potently inhibited by two IP thiol compounds; IP3S3 (1,4,6) and IP3S3 (2,3,5), that show complex binding kinetics (Hill numbers < 1) and can distinguish different forms of the 5-phosphatase in purified membranes. These two inhibitors could be very useful tools to determine the role of the inositol lipid pathway in neuroexcitability.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.