Simulataneous and independent versus antagonistic inhibition of muscle carbonic anhydrase (CA III) by acetazolamide and cyanate

The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO₂ hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO₂ hydration was found to be 6000 sec^?1 with a K_m of 83 mM for CO₂. Cyanate inhibition (K_i, 3 μM) and acetazolamide inhibition (K_i, 44 μM) were both found to be noncompetitive with respect to CO₂. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large K_i for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinant zinc in the catalytic mechanism of CO₂ hydration for CA III.     

The effects of vanadate on rabbit ventricular muscle adenylate cyclase and sodium pump activities

Vanadate in the +5 oxidation state has been reported to have a positive inotropic action on cardiac ventricular muscle. We have investigated the biochemical actions of vanadate on ventricular muscle adenylate cyclase and sodium pump activities in both intact or disrupted cell systems in an attempt to elucidate the mechanism(s) responsible for the physiological response.Vanadate at concentrations up to 100 μM (K_a = 2 μM) stimulated adenylate cyclase activity in sarcolemmal membrane preparations or disrupted myocytes isolated from rabbit ventricular muscle by 2–3-fold. Increasing the vandate concentrations above 100 μM resulted in a progressive inhibition of basal or hormone-stimulated adenylate cyclase activity (K_i = 5 mM) which was similar to that found by the reaction product, pyrophosphate (K_i = 0.5 mM). Both activation and inhibition by vanadate was fully reversible. Maximum activation of adenylate cyclase by vanadate and isoprenaline were not additive whereas maximum fluoride activation was decreased (18%) and the forskolin-stimulated response was slightly potentiated. Vanadate reversibly inhibited ouabain-sensitive p-nitrophenylphosphatase activity (K_i = 60 nM) in sarcolemmal membrane preparations and disrupted myocytes. Complete inactivation was found at 1 μM vanadate.Acute or chronic incubation of intact myocytes with vanadate at concentrations up to 0.5 mM had no measurable affect on ouabain-sensitive ⁸⁶Rb influx or isobutylmethylxanthine, isoprenaline or forskolin-stimulated accumulation of intracellular cAMP concentration. Inhibition of ⁸⁶Rb influx and cAMP accumulation was found at higher concentrations of vanadate; however, this accompanied the progressive decrease in cell viability as measured by the decrease in percentage of rod-shaped cells.It is concluded that vanadate, at concentrations which have been reported to induce a positive inotropic action on mammalian ventricular muscle, does not increase adenylate cyclase activity or inhibit the sodium pump activity in intact myocytes. These results show that caution must be applied when extrapolating the actions found with vanadate in broken cell systems to intact tissues.     

Aspartate carbamoyltrasferase activity,drug concentrations,and pyrimidine nucleotides in tissue from patients treated with N-(phosphonacetyl)-L-aspartate

Biopsy specimens were obtained from patients treated with $\text{N-(}\text{phosphonacetyl)-}\text{L}\text{-aspartate}$ (PALA) in a phase I clinical trial. Activities of aspartate carbamoyltransferase (ACTase), the target enzyme, in ten specimens before treatment varied from 0.4 to 1.7 units/mg. PALA was measured in protein-free extracts of thirteen specimens by inhibition of rat ACTase. At 1.5 to 145 hr after doses of 1 to 6 g/m², PALA concentrations were 0.9 to 89 μg/g; at 4 hr or later the tissue concentrations were similar to those in plasma (five samples). The observed inhibition of ACTase (17–87%) correlated with the PALA concentrations. Pyrimidine nucleotides were decreased (relative to purine nucleotides) in nine of ten specimens, by 16–72%. ACTase partially purified from human spleen had a K_m for carbamoyl phosphate of 20.6 μM and the K_i for PALA was 0.011 μM. The results suggest that inhibition of ACTase by PALA affects the concentration of pyrimidine nucleotides in human tumors in a dose-dependent manner.     

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 cells

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 μM [³H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (K_i = 2.5 μM), 4-biphenylacetic acid (K_i = 5.3 μM), and flurbiprofen (K_i = 5.2 μM), whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (K_i = 0.08 μM), ethacrynic acid (K_i = 0.52 μM), and 1-chloro-2,4-dinitrobenzene (K_i = 0.77 μM). Bilirubin ditaurate was a potent inhibitor of both transport components (K_i = 1.5 and 0.17 μM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 μM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave K_m values of 170 and 250 μM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.     

beta-adrenergic receptors of human leukocytes: studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine

Owing to the large differences in reported values for β-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[³H]dihydroalprenolol (DHA) and $\text{(±)-}\text{[}{}^{125}\text{I]iodohydroxybenzylpindolol}$ (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10–24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000–60,000 sites/cell) of low affinity (K_d 12–15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 ,μM chloroquine not only in PMN cells but also in the lysome-poor MN cells (? 90% lymphocytes), leaving 2000–3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (±)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 μM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent K_d. Mean (± S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 ± 100 sites/MN cell and 1135 ±129 sites/PMN cell (K_d 143–153 pM) using (-)-DHA; and 1487 ± 210 sites/MN cell and 1065 ± 69 sites/PMN cell [avg. K_d(±) 224–274 pM] using (±)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the β₂-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (±)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (±)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[³H]DHA, 1 ,μM (-)-propranolol and 10, μM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for β-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.     

Anti-convulsants and brain aldehyde metabolism: Inhibitory characteristics of ox brain aldehyde reductase

The major isoenzyme of aldehyde reductase has been purified from ox brain by affinity chromatography. Carbamazepine (K_i = 7.3 × 10^?4 M) and phenacemide (K_i = 2.5 × 10^?4 M), in common with all other established anti-convulsant drugs tested, have been shown to inhibit the activity of this enzyme. A selection of structural analogues of the anti-convulsant sodium valproate were found to be potent inhibitors of the reductase (K_ivalues in the range 10^?3 M ?5 × 10^?5 M) and these analogues also showed anti-convulsant activity in the mouse maximal electroshock test. A third group of compounds, the flavonoids, constitute the most potent group of aldehyde reductase inhibitors yet reported. Quercetin and morin exhibited K_i values less than 1 μM. The possible relationship between aldehyde metabolism and anti-convulsant action is discussed and structural characteristics pre-disposing to potent inhibition of aldehyde reductase are described.     

Studies on the reaction catalyzed by transport (Na, K) adenosine triphosphatase. I. Effects of divalent metals

The effects of divalent metals (Cu²⁺, Zn²⁺, Fe²⁺ and Pb²⁺) on a microsomal preparation of NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from beef cerebral cortex were studied. These metals are all potent inhibitors of the enzyme with I₅₀ values of 1 μM for Cu²⁺ and Zn²⁺, 3 μM for Fe²⁺ and 20 μM for Pb²⁺. Kinetic studies examining the effect of low concentrations of divalent metals on K_m and V for MgATP are reported. The results indicate that Fe²⁺ and Pb²⁺ are competitive inhibitors of NaK-ATPase with K_i values of 1.60 μM and 1.90 μM respectively. Cu²⁺ and Zn²⁺ are noncompetitive inhibitors of NaK-ATPase with K_i values of 1.18 μM and 3.48 μM respectively.     

Inhibition of mushroom tyrosinases by lamprene and thiambutosine

The inhibition by lamprene of the oxidation of l-dopa, catalysed by tyrosinase from the wild mushroom, Xerocomus badius, was of the mixed type with a K_i, of 30 μM. The inhibition by thiambutosine of the tyrosinase from the cultivated mushroom, Agaricus campestris, was competitive with a K_i of 15 μM.     

Effects of enprofylline on A1 and A2 adenosine receptors

The effects of enprofylline were studied on A₁ adenosine receptors of rat fat cells and on A₂ adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5′-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a K_B of 130 μM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A₂ receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N⁶-R-(−)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a K_B of 32 μM. The K_i value for inhibition of [³H]PIA binding to fat cell membranes was 45 μM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with K_i values of 15, 130 and 110 μM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A₁ and A₂ adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline.     

The effect of halothane on the stability of Ca2+ transport activity of isolated fragmented sarcoplasmic reticulum

The effects of the inhalation anaesthetic agent, halothane (CF₃CHBrCl), on the stability of the calcium transport system of isolated rabbit white skeletal muscle sarcoplasmic reticulum have been studied. Calcium transport activity was unaffected when suspensions of sarcoplasmic reticulum vesicles were preincubated at 37° and pH 6.8 at concentrations of halothane below 5 mM, but was progressively inactivated at higher concentrations. (Ca²⁺,Mg²⁺)-ATPase activity was enhanced during inactivation of calcium transport. At pH 6.3 and 5.8, halothane increased the first order rate constants of inactivation and effects were noted in the anaesthetic range of concentration (1–2 mM). The inulin inaccessible space of membrane vesicles did not change appreciably during the period of treatment with halothane, excluding increased permeability as an explanation of the inhibition of calcium accumulation. Inactivation was irreversible and highly temperature dependent, with an activation energy of 52.7 kcal/mol. Calcium ions had a protective effect against inactivation (K_{0.5 (Ca2+) = 1.5 × 10^?6M}), as did ATP ($\text{K}{}_{\mathrm{<mtext>0.5\; (</mtext><mtext>Atp</mtext><mtext>)</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$). It is concluded that mild acid conditions and halothane act synergistically during inactivation of the calcium transport system of sarcoplasmic reticulum membranes. These studies suggest that halothane interacts with the (Ca²⁺, Mg²⁺)-ATPase protein at the ATP-specific binding site or that it disrupts protein-lipid associations in the membrane. In either case the destabilizing effect of halothane may be modified by the conformational state of the protein.     

Determination of Protein Binding of Troglitazone Stereoisomers by Fluorometric Titration

Determination of the protein binding of troglitazone is difficult because of its high adsorption to filters and membranes and the instability of the stereoisomers. We attempted to assess the protein binding of four stereoisomers of troglitazone in the plasma and albumin from several species by the method using fluorescent probes. The inhibition constants (K_i) for the stereoisomers of troglitazone were obtained from the decreases in fluorescence intensity of dansylsarcosine caused by competitive inhibition. Each stereoisomer of troglitazone displaced dansylsarcosine, a typical specific fluorescent probe for the diazepam binding site on human serum albumin (HSA). The highest binding affinity for dansylsarcosine was observed with HSA (dissociation constant, K_d,1 = 0·5 μM), while it was lowest in the mouse (K_d,1 = 18 μM). The K_i values for KK and ddY mouse plasma and mouse and rat albumin were in the range 2–15 μM, and there were no large variations among stereoisomers, the maximum differences being twofold. For human plasma and albumin, the displacement could not be accounted for by a simple competitive inhibition. Comparison between unbound fraction ( f_u) values calculated from thus obtained K_i values and those of a mixture of the four stereoisomers determined by high-performance frontal analysis showed that the f_u values obtained by fluorometric titration were higher, while the relative differences among the stereoisomers in terms of animal species and strain were comparable for the two methods. Small differences in protein binding among stereoisomers of troglitazone may not be the major reason for their stereoselective pharmacokinetics. © 1997 John Wiley & Sons, Ltd.     

Effect of synthetic purines and purine nucleosides on [3H]diazepam binding in brain

We have compared fifteen synthetic purines and purine nucleosides on their ability to displace [³H]diazepam binding to rat brain membranes. Among these analogs, 6-methylthioguanine was found to be most potent, inhibiting competitively the specific binding of [³H]diazepam with a K_i value of 16 μM. At a concentration of 50 μM, 6-methyl-thioguanine increased the apparent K_D of specific diazepam binding from 4.3 nM to 13.3 nM without affecting the B_max, nor had it any effect on the non-specific binding. Binding with membrane preparations from developing rat brain was slightly less sensitive to 6-methylthioguanine inhibition than that with membranes prepared from mature brain.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Inhibitory effects of class I and IV antiarrhythmic drugs on the Na+-activated K+ channel current in guinea pig ventricular cells

Recently we have reported that class III antiarrhythmic drugs including amiodarone inhibit the Na⁺-activated K⁺ (K_Na) channels in isolated cardiac cells. In this study effects of antiarrhythmic drugs having class I and/or IV properties on the single K_Na channel current were examined in inside-out membrane patches of guinea pig ventricular cells by using patch clamp techniques. The K_Na channel current, which was activated by increasing [Na⁺]_i from 0 mM to 100 mM in the presence of 150 mM [K⁺]_o, showed a large slope conductance (212 pS) and inward-going rectification. Quinidine (100 μM), mexiletine (100 μM) and flecainide (10 μM) were selected as representative of class Ia, Ib and Ic drugs, respectively. These drugs at relatively high concentrations incompletely inhibited the K_Na channel by decreasing the open time (flickering block). The class IV drug verapamil inhibited the K_Na channel current mainly by decreasing the open probability although the IC₅₀ value of verapamil (3.36 μM) was higher than the therapeutic concentrations. Bepridil and SD-3212, antiarrhythmic drugs having both class I and IV properties, potently inhibited the K_Na channel current by decreasing the open probability. The IC₅₀ values of bepridil and SD-3212 for inhibiting the K_Na channel current was 0.51 μM and 0.53 μM, respectively, both of which are within the therapeutic range. Most antiarrhythmic drugs inhibit cardiac K_Na channels by different modes and at different concentrations. The K_Na channel blocking action of bepridil and SD-3212 may partly contribute to the prolongation of the action potential duration by these drugs at rapid stimulation rates. Received: 20 April 1998 / Accepted: 16 September 1998     

The inhibition of catechol-O-methyltransferase by 2,3-dihydroxypyridine

Despite its structural similarity to catechol, 2,3-dihydroxypyridine is not a substrate but a “dead-end” inhibitor of purified pig liver catechol-O-methyltransferase. It inhibits the methylation of 3,4-dihydroxyphenylacetic acid competitively with an inhibitor constant of 15 μM. Against the methyl donor, $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$, it is an uncompetitive inhibitor (K′_i = 85 μM). Clearly, although 2,3-dihydroxypyridine interacts with the catechol-binding site of the enzyme, the presence of a nitrogen in the ring alters its susceptibility to O-methylation.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

Transport and interaction of drugs in leukocytes

The accumulation of selected CNS drugs by rat leukocytes was previously reported. This paper presents evidence for the transport into leukocytes of additional drugs. Also studied was the inhibition of the latter processes by various structurally related compounds. The markedly rapid and sodium-independent uptakes into rat leukocytes of amphetamine, codeine, methadone and naloxone fulfilled the basic criteria for active transport. The uptake of morphine was apparently accomplished by more than one process. The affinities of the high capacity transport systems (approximate V_max: 100 nmoles/g cells/5sec) varied considerably as reflected by the two extreme K_m values obtained for methadone (20 μM) and morphine (1.8 mM). A variety of amines inhibited the cellular transport of the drugs. Most potent inhibitors were quinacrine (K_i: 0.5 to 3 μM), desipramine (K_i: 6–20 μM) and methadone (K_i: 18–25 μM). Morphine and tryptamine exhibited inhibition constants higher than 1 mM. The cellular transport processes newly described in rat leukocytes apparently represent a novel addition to the heterogenous biological transport of basic amines. The structural specificity of amine transport in various tissues is discussed.     

Patent Evaluation: The Use of Calpain Inhibitors in the Treatment of Neurodegeneration

Summary

Novelty: Novel calpain inhibitors, including ketopeptides, halopeptides and isocoumarins are disclosed. They are said to be useful in the treatment of neurodegenerative diseases due to their effect on intracellular free calcium. Examples of use include stroke, Alzheimer's disease, head trauma and HIV-induced neuropathy.

Biology: Comprehensive data are presented, including K_i values for the inhibition of cysteine proteases (papain, (P); calpain I, (CI); cathepsin B, (CB); and calpain II, (CII)). Two peptide examples are Z-Leu-Phe COOEt (K_i CI = 0.23 and CII = 0.4 μM) and Z-Leu-Abu-COOEt (K_i CI = 0.04 and CII = 0.4 μM). An example of the isocoumarins is 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin (IC₅₀ CI = 10 μM). A figure is given showing inhibition of glutamate-induced cell death by a number of calpain inhibitors.

Chemistry: A very large number of ketopeptides and isocoumarins are exemplified. The peptides are prepared using standard techniques.

Structure:      

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

In situ characterization of nasal leucine enkephalin degrading aminopeptidase susceptibility of the nasal enzyme to boronic acids and phosphorus-containing peptide and amino acid isosteres

The N-terminal Tyr-Gly bond of leucine-enkephalin is specifically hydrolyzed during exposure to the nasal mucosa. Kinetic properties in situ indicate that this activity is due to a single enzyme which has a K_M^app of 0.4 mM for leucine-enkephalin. Analysis of initial rates of hydrolysis from earlier inhibition studies using boroalanine, boroleucine, and borovaline indicated that these inhibitors bind the nasal enzyme with K_m^app values of 0.009–0.02 μM. In addition, we have evaluated borophenylalanine (K^app_i = 0.004 μM) in this study. Similarly, H-PheΨP(O)(OH)CH₂Phe-OMe binds the nasal aminopeptidase with a K^app_i of 0.2 μM. Comparison of these K_i values with those of cytosolic aminopeptidase and microsomal aminopeptidase derived from porcine kidney, indicates that the nasal enzyme closely resembles the microsomal enzyme in properties. Major distinctions between the enzymes are: (1) the greater dependence of the cytosolic enzyme on the nature of the amino acid residue in the primary site (2) a much greater preference of both the microsomal and nasal enzyme for HPheΨ[P(O)(OH)CH₂]Phe-OMe over H-PheΨ[P(O)(OH)₂].