Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10–200 μM) stimulation of 5-HT transport that was rapid and sustained over 0–10 minutes of incubation. When platelets were incubated with diamide (10–200 μM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 μM, while washed platelets incubated with diamide (10–200 μM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (V_max control = 28.4 ± 1.4 vs. V_max diamide = 60.9 ± 4.1 pM/10⁸ platelets/4 min) but did not significantly alter the K_m values. Ouabain, an inhibitor of platelet Na⁺-K⁺ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.     

Surface-associated glycosyltransferase activities in rat sertoli cells in vitro

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a V_max of 12.5 pmoles/mg protein/min and a K_m of 22 μM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a V_max of 10 pmoles/mg protein/min and a K_m of 18.2 μM for GDP-[¹⁴C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a V_max of 50 pmoles/mg protein/min and a K_m of 38.5 μM, while SCPM showed GalTase activity with a V_max of 25 pmoles/mg protein/min and a K_m of 20.8 μM for UDP-[³H]-galactose. Galactosyltransferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, ～33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells. These results suggest that cell surface glycosyltransferases may be involved in Sertoli cell function during mammalian spermatogenesis. © 1993 Wiley-Liss, Inc.     

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Vinyl functionalized 5,6-dimethylbenzimidazolium salts: Synthesis and biological activities

A series of vinyl functionalized 5,6-dimethylbenzimidazolium salts are synthesized. All compounds were fully characterized by elemental analyses, MS, ¹H-NMR, ¹³C-NMR, and IR spectroscopy techniques. Enzyme inhibition is a very active area of research in drug design and development. In this study, the synthesized novel benzimidazolium salts were evaluated toward the human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. They demonstrated highly potent inhibition ability against hCA I with K_i values of 484.8 ± 62.6–1389.7 ± 243.2 nM, hCA II with K_i values of 298.9 ± 55.7–926.1 ± 330.0 nM, α-glycosidase with K_i values of 170.3 ± 27–760.1 ± 269 μM, AChE with K_i values of 27.1 ± 3–77.6 ± 1.7 nM, and BChE with K_i values of 21.0 ± 5–61.3 ± 15 nM. As a result, novel vinyl functionalized 5,6-dimethylbenzimidazolium salts (1a–g) exhibited effective inhibition profiles toward studied metabolic enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly to treat some global disorders including glaucoma, Alzheimer's disease, and diabetes.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

Influence of the time interval after venepuncture and the storage temperature on the uptake of 14C-serotonin by human blood platelets

The active uptake of ¹⁴C-5-HT into human platelets at 37°C was studied at various times (10–130 min) and at various storage temperatures (4°, 22°, 37°C) after venepuncture. 5-HT uptake was significantly influenced by both variables. There was no direct correlation between 5-HT uptake and storage temperatures, storage time and changes in the pH of PRP, resp. The apparent K_m-value for the 5-HT uptake (0.5μM) remained constant. However, the K_i-values obtained for different uptake inhibitors at the different experimental conditions indicate the need for exact standardization.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 ± 0.06 μmol/g vs. 3.84 ± 0.13 μmol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (V_max) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase V_max increased from 146 ± 29 to 401 ± 94 nmol/min per g; substrate K_m values of neither enzyme were affected by diabetes. Net glutamine efflux (AZ concentration difference × blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (?30.0 ± 3.2 vs. ?1.9 ± 2.6 nmol/min per g (P < 0.001) and hindlimb NH₃ uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (V_max) of the Na-dependent System N^m in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 ± 50 vs. 1160 ± 80 nmol/min per g (P < 0.01)), but transporter K_m was the same in both groups (9.2 ± 0.5 nM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. In Intramuscular Na⁺ was 26% greater in diabetic (13.2 μmol/g) than control muscle, but muscle K⁺ (100 μmol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of; (i), a diminished Na⁺ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased V_max/K_m for sarcolemmal glutamine efflux.     

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (?) ³H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K_D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (?)Isoproterenol (10^?5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (?)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-endorphin receptor the decapeptide immunorphin ([³H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [³H]immunorphin binds with a high affinity to the non-opioid β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-endorphin were synthesized, and their ability to inhibit the specific binding of [³H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

Novel 12-hydroxydehydroabietylamine derivatives act as potent and selective butyrylcholinesterase inhibitors

The skeleton of the diterpene dehydroabietylamine was modified, and a set of 12-hydroxy-dehydroabietylamine derivatives was obtained. The compounds were screened in colorimetric Ellman’s assays to determine their ability to act as inhibitors for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum). Additional investigations concerning the enzyme kinetics were performed and showed 12-hydroxy-N-(4-nitro-benzoyl)dehydroabietylamine (13) and 12-hydroxy-N-(isonicotinoyl)dehydroabietylamine (17) as selective BChE inhibitors holding good inhibition constants K_i = 0.72 ± 0.06 μM and K_i = 0.86 ± 0.19 μM, respectively.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

The Uptake of Pyrimidine Nucleosides in Tetrahymena. I. Uridine*

SYNOPSIS. Uridine uptake was examined in Tetrahymena pyriformis GL-7 in defined medium under conditions where food vacuole formation is not a significant factor in solute acquisition by the cell. The results indicate the presence of a saturable mechanism which follows Michaelis-Menten kinetics. When corrected for diffusion the apparent K_m for the carrier is 2.3 ± 0.6 μM and the V_max is 7.3 ± 0.2 × 10^?7 nmoles/cell/min. It is evident from nucleotide pool analysis that most of the radioactivity of externally supplied [³H]uridine appears in UMP with the remainder in UTP. Uridine is apparently phosphorylated immediately upon entry into the cell and neither uridine-cytidine kinase activity nor RNA synthesis are rate-limiting in the uptake process. Uridine transport is competitively inhibited by a variety of ribo- and deoxyribonucleosides as well as several nucleoside analogs. Neither uracil nor ribose or deoxyribose are effective inhibitors of uridine transport indicating the carrier is specific for the nucleoside. There is little difference between the K_i values for ribo- as opposed to deoxyribonucleosides except in the case of deoxyguanosine which is much less effective as an inhibitor under the conditions of this study, than all the other nucleosides, including guanosine.