Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.     

Inhibition and activation of porcine squalene epoxidase.

Pig liver squalene epoxidase (SE) has been partially purified from solubilized microsomes by DEAE-Sephacel and Blue Sepharose 4B chromatography. This stable and reproducible preparation was used to investigate the mechanism of several substrate-like inhibitors of SE and to study the effects of pH, metals, detergents, and cofactors on enzyme activity. Most divalent (1 mM) and trivalent (0.1 mM) metal cations had little effect on SE at pH 7.4; only ferrous and cupric ions showed ca. 50% reduction in SE activity. Interestingly, at pH 8.8, EDTA (10 mM) shows 1.8-fold enhancement of enzyme activity. Among the detergents, Triton X-100 was clearly superior for solubilization and purification of porcine SE; Tween 80, Lubrol-PX, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, octyl beta-glucoside, and three different Zwittergents were much less effective for SE solubilization. Partially purified pig liver SE showed maximal activity at pH 8.8-9.0. Trisnorsqualene alcohol and trisnorsqualene cyclopropylamine were noncompetitive inhibitors at pH 8.8, with Ki values of 4 microM and 180 nM, respectively; these two inhibitors were not substrates for SE. In contrast, 26-hydroxysqualene was both a competitive inhibitor with a Ki value of 4 microM at pH 8.8 and a substrate for SE. An unexpected enhancement (up to 350%) of SE activity was observed at pH 7.4 following preincubation with selected nonpolar derivatives of farnesol and farnesoic acid. At pH 8.8, this effect was less dramatic but still evident.     

Adenosine deaminase activity of chicken erythrocyte and heart cytosols

1. The adenosine deaminase (ADA) activities of chicken erythrocyte and heart cytosols had pH optima of 6.5. The temperature optima for erythrocyte and heart ADA were 30 and 35 degrees C, respectively. 2. The deoxyadenosine/adenosine deamination ratios ranged from 0.75 to 0.84 for both ADA activities. 3. For erythrocyte ADA, Km values were 8.9-12.9 microM adenosine (range) and 8.3 microM 2'-deoxyadenosine. For heart ADA, Km values were 6.7-12.0 microM adenosine (range) and 5.3 microM 2'-deoxyadenosine. 4. Inosine was a competitive inhibitor of both erythrocyte (Ki = 73 microM) and heart (Ki = 109 microM) ADA.     

New renin inhibitors homologous with pepstatin.

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.     

Inhibition of acetoacetyl-CoA synthetase from rat liver by fatty acyl-CoAs

The activity of acetoacetyl-CoA synthetase from rat liver was found to be negatively regulated by coenzyme A, fatty acyl-CoAs and acetoacetyl-CoA in vitro. With increasing concentrations of coenzyme A (substrate inhibition occurring at concentrations higher than 50 microM) the pH optimum shifted toward the acidic side (7.5-8.5 with 5 microM coenzyme A and 6.5-7.0 with 500 microM coenzyme A), in parallel with progressively decreasing enzyme activity. Fatty acyl-CoAs of various chain lengths dose-dependently inhibited acetoacetyl-CoA synthetase from rat liver, but much less effectively a similar enzyme from a bacterium, Zoogloea ramigera I-16-M. Palmitoyl-CoA, the most potent inhibitor of the rat liver enzyme, with an apparent Ki value of 9.8 microM, apparently inhibited the enzyme below its critical micellar concentration, not due to its detergent action. Acetoacetyl-CoA showed product inhibition with a Ki value of 15 microM. These results suggest a possible physiological regulation mechanism for this enzyme with respect to fatty acid biosynthesis.     

Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids.

The hydroxamic acid HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe, isomer 6A (GM 6001), inhibits human skin fibroblast collagenase with Ki of 0.4 nM using the synthetic thiol ester substrate Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt at pH 6.5. The other isomer, 6B, which has the opposite configuration at the CH2CH(i-Bu)CO alpha-carbon atom, has a Ki of 200 nM for this enzyme. GM 6001 is one of the most potent inhibitors of human skin fibroblast collagenase yet reported. GM 6001 has a Ki of 20 nM against thermolysin and Pseudomonas aeruginosa elastase. Isomer 6B has a Ki of 7 nM against thermolysin and 2 nM against the elastase. 6A and 6B are the most potent hydroxamate inhibitors reported for these bacterial enzymes. The pattern of inhibition for all three enzymes suggests that isomer 6A is the (R,S) compound, stereochemically analogous to the L,L-dipeptide, and isomer 6B is the (S,S) compound, analogous to the DL-dipeptide. The tolerance of the D configuration by thermolysin and the elastase allows these inhibitors to discriminate between the human and bacterial enzymes simply by inversion of configuration at the CH2CH(i-Bu)CO alpha-carbon atom. Substitution of the potential metal liganding groups carboxylate and hydrazide for the hydroxamate group yields much weaker inhibitors for all three enzymes.     

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.     

The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera

The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase. Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.     

Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii

Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii. It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S. The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group. pCMB inhibited the enzyme activity (IC50 = 2 microM). The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C. It is activated by 0.1 M KCl and KI and 2 M urea. 3-4 M urea completely inactivated the enzyme. Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.     

Steady-state magnesium ion-adenosine triphosphatase activities of myosin and its subfragment 1 derivative

The Mg2+-ATPase activity of myosin and its subfragment 1 (ATP phosphohydrolase, EC 3.6.1.3) always followed normal Michaelis-Menten kinetics for ATP concentrations less than 10 microM. The average Km values at pH 7.4 and 25 degrees C are 0.33 +/- 0.04 microM for myosin and 0.43 +/- 0.11 microM for subfragment 1. At low salt concentration myosin yields a second hyperbolic increase in Mg2+-ATPase activity as the ATP rises from 10.2 microM to 153 microM: V doubles with a Km of 11 +/- 5 microM. This second low-salt-dependent increase in Mg2+-ATPase activity occurred between pH 6.8 and pH 8.7. It was not affected by the presence of 0.10 M EGTA to remove Ca2+ contamination. Solubilization of the catalytic sites by assaying myosin for ATPase activity in the presence of 0.60 M NaCl or by conversion of myosin to subfragment 1 eliminated the secondary hyperbolic increase. Subfragment 1 has a significantly different pH-activity curve from that of myosin. Subfragment 1 has an activity peak at pH 6.0, a rising activity as the pH goes from 8.7 to 9.8, and a deep activity valley between pH 6.8 and pH 8.4. Myosin has a very shallow trough of activity at pH 6.8 to 8.4, and in 1.0 mM ATP its activity drops as the pH decreases from 6.8 to 6.0. NaCl is a noncompetitive inhibitor of the Mg2+-ATPase activity of myosin and subfragment 1. Myosin has a greater affinity for NaCl (Ki = 0.101 +/- 0.004 M) than does subfragment 1 (Ki = 0.194 +/- 0.009 M).     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

Angiotensin I converting enzyme inhibitors containing unnatural alpha-amino acid analogues of phenylalanine

The activity of three angiotensin I converting enzyme (ACE) inhibitors with unique related structures was assessed in vitro and in vivo. The three compounds were (S)(-)-1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-3-isoquinoline carboxylic acid (EU-4865), 1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-1- isoquinolinecarboxylic acid (EU-4881), and (S)(-)-1,2,3,4-tetrahydro-1-(3-mercapto-1-oxopropyl)-2- quinolinecarboxylic acid (EU-5031). In vitro EU-4881 was a competitive inhibitor that lacked potency (IC50 = 1980 nM) against purified ACE. The other two compounds were equipotent (IC50 = 41 nM) against purified ACE but differed in their inhibition kinetics. EU-4865 (Ki = 38 nM) was a noncompetitive inhibitor, and EU-5031 (Ki = 6.9 nM) was a competitive inhibitor. Against caveolae membrane-bound ACE EU-4881 also lacked potency (IC50 = 2852 nM). In vivo in the conscious acute aortic coarctate (AAC) rat it also lacked potency, having an ED30 (oral dose decreasing blood pressure 30 mmHg) greater than 100 mg/kg. The activity of EU-4865 and EU-5031 in the caveolae membrane-bound ACE and AAC rat reflected their different Ki values rather than their similar IC50 values. In vitro, EU-4865 and EU-5031 had IC50 values of 19 and 6.7 nM, respectively, and in vivo, they had ED30 values of 52 and 1.1 mg/kg, respectively. These results suggest that ACE has a binding requirement for a carboxy-terminus, hydrophobic amino acid that is important for in vivo activity.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.     

Kinetics of enzymic reductive deiodination of iodothyronines. Effect of pH.

5'-Deiodination of thyroxine (yielding 3,3',5-tri-iodothyronine; reaction I) and of 3,3',5'-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction II) and 5-deiodination of thyroxine (yielding 3,3',5'-tri-iodothyronine; reaction III) and of 3,3',5-tri-iodothyronine (yielding 3,3'-di-iodothyronine; reaction IV) as catalysed by rat liver microsomal fraction were studied at pH 6.5, 7.2 and 8.0 It was found that: (1) the Km of reaction I was relatively independent of pH (approx. 3 microM), whereas V was highest at pH 6.5 (63 pmol of 3,3',5-tri-iodothyronine/min per mg of protein); (2) the Km of reaction II was lowest at pH 6.5 (0.035 microM), but V was highest at pH 8.0 (829 pmol of 3,3'-di-iodothyronine/min per mg of protein); (3) thyroxine inhibited reaction II competitively; Ki values were identical at pH 6.5 and 8.0 (1 microM); (4) for both reactions III and IV Km was lowest and V was highest at pH 8.0. The results are compatible with the view that reactions I and II are mediated by a single enzyme (iodothyronine 5'-deiodinase) and that reactions III and IV are catalysed by a second enzyme (iodothyronine 5-deiodinase).     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The antiheparin effect of a heparinoid, pentosan polysulphate. Investigation of a mechanism.

A pentosan polysulphate [a fully sulphated (1-4)-beta-D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid] has been shown to inhibit the anticoagulant activity of high-affinity heparin as observed in plasma and when using purified enzyme and inhibitor. The activity was shown to be concentration-dependent with an apparent Ki of approx. 2 microM. The antiheparin property was not shown by a number of other anionic carbohydrates when tested. The rate of thrombin inhibition at 0.33 microM-heparin was reduced from 7.1 X 10(8) M-1 X min-1 in the absence of pentosan polysulphate to 2.3 X 10(8) M-1 X min-1 at 2 microM-pentosan polysulphate and to 0.3 X 10(8)M-1 X min-1 at 20 microM. Using the random bireactant model of heparin action [Griffiths (1982) J. Biol. Chem. 257, 13899-13902] it was observed that the pentosan polysulphate had no effect on the Km for antithrombin III (150 nM) but increased the Km for thrombin from 25 nM to 450 nM. A reduction in the inhibition rate by 17.3-fold predicted by substitution of these values into the general two-substrate reaction-rate equation was confirmed experimentally.     

Isolation and purification of a neutral alpha(1,2)-mannosidase from Trypanosoma cruzi

Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans. Although a fair amount is known about the biochemistry of certain trypanosomes, very little is known about the enzymic complement of synthesis and processing of glycoproteins and/or functions of the subcellular organelles in this parasite. There have been very few reports on the presence of acid and neutral hydrolases in Trypanosoma cruzi. Here we report the first purification and characterization of a neutral mannosidase from the epimastigote stage of Trypanosoma cruzi. The neutral mannosidase was purified nearly 800-fold with an 8% recovery to apparent homogeneity from a CHAPS extract of epimastigotes by the following procedures: (1) metal affinity chromatography on Co+2-Sepharose, (2) anion exchange, and (3) hydroxylapatite. The purified enzyme has a native molecular weight of 150-160 kDa and is apparently composed of two subunits of 76 kDa. The purified enzyme exhibits a broad pH profile with a maximum at pH 5.9-6.3. It is inhibited by swainsonine (Ki, 0.1 microM), D-mannono-delta-lactam (Ki, 20 microM), kifunensine (Ki, 60 microM) but not significantly by deoxymannojirimycin. The enzyme is activated by Co2+and Ni2+and strongly inhibited by EDTA and Fe2+.The purified enzyme is active against p-nitrophenyl alpha-D-mannoside (km = 87 microM). High-mannose Man9GlcNAc substrate was hydrolyzed by the purified enzyme to Man7GlcNAc at pH 6.1. The purified enzyme does not show activity against alpha1,3- or alpha1,6-linked mannose residues. Antibodies against the recently purified lysosomal alpha-mannosidase from T.cruzi did not react with the neutral mannosidase reported here.     

Purification and characterization of enkephalin-degradating enzymes from calf-brain striatum

Enkephalinase A and B are extracted from Triton-X 100 washed calf-brain particles and purified by DEAE-cellulose chromatography. Both enzymes have identical Km values in their membrane-bound and soluble form. Enkephalinase A has a pH optimum at 6.9 and a Km for Leu-enkephalin of 20-25 microM, which hardly depends on the pH. Thiorphan and phosphate are purely competitive inhibitors of Enkephalinase A with Ki values of 3 nM and 1.5 mM respectively (pH = 6.85). Enkephalinase B is not affected by phosphate or thiorphan. It has a Km for Leu-enkephalin of 10 microM, a pH optimum of 7.0 and is inhibited by low concentrations of apolar dipeptides.     

Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.     

Inhibition of angiotensin converting enzyme by L-alanyl-4 or 5-substituted-L-prolines and their N alpha-phosphoryl-derivatives

A series of L-alanyl-4 or 5-substituted L-prolines, such as L-alanyl-L-thiazolidine-4-carboxylic acid, L-alanyl-5-oxo-L-proline, L-alanyl-trans-4-hydroxy-L-proline and L-alanyl-cis-4-hydroxy-L-proline as well as their corresponding N alpha-phosphoryl derivatives, were synthesized and studied for inhibition against angiotensin converting enzyme. Furanacryloyl-phenylalanyl-glycyl-glycine was used as substrate in 50 mM Tris hydrochloride buffer at pH 7.5 containing 1 microM zinc acetate. N alpha-Phosphoryl-L-alanyl-L-thiazolidine-4-carboxylic acid and N alpha-phosphoryl-L-alanyl-trans-4-hydroxy-L-proline competitively inhibit angiotensin converting enzyme with Ki values of 68 microM and 89.3 microM, respectively. Smaller inhibition against angiotensin converting enzyme was obtained with the rest of compounds studied here.