Complex Pattern Of Inhibition Research Articles

The interactions of metal cations and oxyanions with protein tyrosine phosphatase 1B

Protein tyrosine phosphatases are not considered to be metalloenzymes. Yet, they are inhibited by zinc cations and metal and non-metal oxyanions that are chemical analogues of phosphate, e.g. vanadate. Metal inhibition is generally not recognized as these enzymes are purified, supplied, and assayed with buffers containing chelating and reducing agents. We screened a series of cations and anions for their capacity to inhibit protein tyrosine phosphatase 1B and discuss the ensuing general issues with inhibition constants reported in the scientific literature. In contrast to zinc, which binds to the phosphocysteine intermediate in the closed conformation of protein tyrosine phosphatase 1B when the catalytic aspartate has moved into the active site, other divalent cations such as cadmium and copper may also bind to the enzyme in the open conformation. Inhibition by both anions and cations, conditions such as pH, the presence of metal ligands such as glutathione, and the existence of multiple conformational states of protein tyrosine phosphatases in the reaction cycle establish a complex pattern of inhibition of these important regulatory enzymes with implications for the physiology, pharmacology and toxicology of metal ions.

A facile method to determine intrinsic kinetic parameters of ω-transaminase displaying substrate inhibition

It is usually time-consuming to determine intrinsic kinetic parameters of bisubstrate enzymes, especially when experimental kinetic data deviate from a linear Lineweaver-Burk plot due to complex inhibition patterns. A typical example is ω-transaminase (ω-TA) which is an industrially important enzyme for asymmetric synthesis of chiral amines. ω-TA catalyzes transfer of an amino group between a donor (D) and an acceptor (A) via a ping-pong bi-bi mechanism and often displays substrate inhibitions by reactive amino acceptors, which leads one to prefer to determine apparent kinetic parameters rather than intrinsic ones despite limited applicability for precise understanding of enzyme properties. Here, we developed a new method to determine intrinsic kinetic parameters of ω-TA by double-reciprocal analysis using only two sets of kinetic data. First, linear regression of 1/initial rate (vi) against 1/[A] was carried out with one set of kinetic data measured at a fixed [D] while [A] lay far below the concentration range under the influence of substrate inhibition. Second, another linear regression of 1/[D] vs 1/vi was conducted with one set of kinetic data obtained at a fixed [A] within a substantial substrate inhibition range. The resulting four equations obtained from the y-intercepts and slopes of the two regression lines were used for determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.

Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

Functional Organization of the Thalamic Input to the Thalamic Reticular Nucleus

Most axons connecting the thalamus and cortex in both directions pass through the thalamic reticular nucleus (TRN), a thin layer of GABAergic cells adjacent to the thalamus, and innervate neurons there. The TRN, therefore, is in a strategic location to regulate thalamocortical communication. We recorded neurons of the somatosensory region of the TRN in a thalamocortical slice preparation and studied the spatial organization of their thalamic input using laser scanning photostimulation. We show that the thalamoreticular pathway is organized topographically for most neurons. The somatosensory region of the TRN can be organized into three tiers. From the inner (thalamoreticular) border to the outer, in a manner roughly reciprocal to the reticulothalamic pathway, each of these tiers receives its input from one of the somatosensory relays of the thalamus--the posterior medial, ventroposterior medial, and ventroposterior lateral nuclei. What is surprising is that approximately a quarter of the recorded neurons received input from multiple thalamic regions usually located in different nuclei. These neurons distribute evenly throughout the thickness of the TRN. Our results, therefore, suggest that there exist a subpopulation of TRN neurons that receive convergent inputs from multiple thalamic sources and engage in more complex patterns of inhibition of relay cells. We propose these neurons enable the TRN to act as an externally driven "searchlight" that integrates cortical and subcortical inputs and then inhibits or disinhibits specific thalamic relay cells, so that appropriate information can get through the thalamus to the cortex.

Characterization of the Metabolic Interaction between Trihalomethanes and Chloroacetic Acids using Rat Liver Microsomes

The aim of this study was to investigate the in vitro metabolism of trihalomethanes (THMs) in the presence of trichloroacetic acid (TCA), dichloracetic acid (DCA), monochloroacetic acid (MCA), and 4-methylpyrazole (4-MP) using liver microsomes from male Sprague-Dawley rats. Using the vial equilibration technique, initial experiments were carried out with starting concentrations of approximately 40 ppm THMs and 12–22 mMchloroacetic acids.The results indicated a mutual metabolic inhibition between THMs present as binary or quaternary mixtures. Although DCA and MCA had no influence on THMs, TCA produced a marked inhibition of the metabolism of all THMs: chloroform (CHCl3) (55%), bromodichloromethane (BDCM) (34%), dibromochloromethane (DBCM) (30%), and bromoform (TBM) (23%). The presence of 4-MP also reduced THM metabolism, the importance of which decreased in the following order: CHCl3 > BDCM > DBCM = TBM. In further vial equilibration experiments, using 9–140 ppm as starting concentrations of THMs, enzyme kinetic parameters (i.e., Michaelis constant, K m, and maximum velocity, V max) were determined both in the absence and in the presence of TCA (12.2 mM). Results are consistent with a competitive inhibition between TCA and CHCl3, whereas the metabolic inhibition of BDCM and TMB by TCA was non-competitive. As for DBCM, results suggest a more complex pattern of inhibition. These results suggest that CYP2E1 is involved in the metabolism of THMs as well as in the metabolic interaction between THMs and TCA. We thank Mylène Beaudoin for her excellent technical assistance during this study. This work was supported by the Toxic Substances Research Initiative (TSRI) of Health Canada and Environment Canada

Inhibition of Bovine Phenol Sulfotransferase (bSULT1A1) by CoA Thioesters: EVIDENCE FOR POSITIVE COOPERATIVITY AND INHIBITION BY INTERACTION WITH BOTH THE NUCLEOTIDE AND PHENOL BINDING SITES

Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J. D. (1999) Biochem. Biophys. Res. Commun. 261, 815-819). Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs. A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate. Convex plots of apparent K(m)/V(max) versus [palmitoyl-CoA] were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme. Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase. Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments. First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner. Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol. Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9). Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain.

Shapes and level tolerances of frequency tuning curves in primary auditory cortex: quantitative measures and population codes.

The shape and level tolerance of the excitatory frequency/intensity tuning curves (eFTCs) of 160 cat primary auditory cortical (A1) neurons were investigated. Overall, A1 cells were characterized by tremendous variety in eFTC shapes and symmetries; eFTCs were U-shaped ( approximately 20%), V-shaped ( approximately 20%), lower-tail-upper-sharp ( approximately 15%), upper-tail-lower-sharp (<2%), slant-lower ( approximately 10%), slant-upper (<3%), multipeaked ( approximately 10%), and circumscribed ( approximately 20%). Quantitative analysis suggests that eFTC are best thought of as forming a continuum of shapes, rather than falling into discrete categories. A1 eFTCs tended to be more level tolerant than eFTCs from earlier stations in the ascending auditory system as inferred from other studies. While individual peaks of multipeaked eFTCs were similar to single peaked eFTCs, the overall eFTC of multipeaked neurons (spanning the range of all peaks) tended to have high-frequency tails. Measurements of shape and symmetry indicate that A1 eFTCs, on average, tended to have greater area on the low-frequency side of characteristic frequency (CF) than on the high-frequency side. A1 cells showed a relationship between CF and the inverse slope of low-frequency edges of eFTCs, but not for high-frequency edges. These data demonstrate that frequency tuning, particularly along the eFTC low-frequency border, sharpens along the lemniscal pathway to A1. The results are consistent with studies in mustached bats (Suga 1997) and support the idea that spectral decomposition along the ascending lemniscal pathway up to A1 is a general organizing principle of mammalian auditory systems. Altogether, these data suggest that A1 neurons' eFTCs are shaped by complex patterns of inhibition and excitation accumulating along the auditory pathways, implying that central rather than peripheral filtering properties are responsible for certain psychophysical phenomena.

Complex pattern of inhibition by Mg 2+ of exocytosis from permeabilised eosinophils

Inhibition by Mg 2+ ions of exocytotic secretion from permeabilised eosinophils, stimulated by Ca 2+ and GTPγS, and in the presence and absence of ATP, has been examined. While Mg 2+ inhibits release of aryl sulphatase, hexosaminidase and peroxidase, we found no evidence that this occurs by competition at a Ca 2+-binding site. On the other hand, the IC 50 for Mg 2+ approximates a simple inverse relationship to EC 50 for GTPγS over a wide range of concentrations, indicative of a possible competition with events directly controlled by a GTP-binding protein. However, for secretion stimulated by GTPγS in the absence of Ca 2+ (which necessitates provision of ATP), the effect of Mg 2+ becomes biphasic. Initially, secretion is dependent on the presence of Mg 2+ as a component of the complex ligand Mg.ATP. At high concentrations, Mg 2+ inhibits secretion and the IC 50 was found to be fixed at a concentration of about 8 mM regardless of the strength of the stimulus. The presence of ATP appears to divert the site of inhibition due to Mg 2+.

Calmodulin N-Methyltransferase: KINETICS, MECHANISM, AND INHIBITORS

The present study was undertaken to determine kinetic and inhibition parameters and the mechanism of S-adenosyl-L-methionine:calmodulin-L-lysine N6-methyltransferase (EC 2.1.1.60, CLNMT), an enzyme for which calmodulin is a substrate. Partially purified CLNMT isolated from rat testes had a Vmax of 540 pmol/min/mg and Km values for mushroom demethylcalmodulin and S-adenosyl-L-methionine of 230 nM and 2.0 microM, respectively. Kinetic analysis indicated a complex Bi Bi sequential kinetic mechanism for CLNMT where S-adenosyl-L-methionine binds initially and is followed by demethylcalmodulin binding. When the effects of 20 different compounds that are either inhibitors of calmodulin-specific or methylation-specific functions were examined, CLNMT displayed a pattern of inhibition which differs from that seen with calmodulin-activated enzymes. The product of calmodulin methylation, fully trimethylated calmodulin, and nonmethylatable VU-3 calmodulin acted as competitive inhibitors of CLNMT, with Ki values of 310 and 400 nM, respectively. Of the 13 compounds tested, which are inhibitors of calmodulin-dependent cyclic nucleotide phosphodiesterase, only the calmodulin-binding domain from Ca2+/calmodulin-dependent kinase II, melittin, and calmidazolium were effective inhibitors of CLNMT and each exhibited a complex pattern of inhibition with Kis values of 21, 50, and 65 nM, respectively. The only potent methylation-specific inhibitor was S-adenosyl-L-homocysteine, which also displayed a complex pattern of inhibition.

Acyl-Coenzyme A:Cholesterol O -Acyltransferase Is Not Identical to Liver Microsomal Carboxylesterase

Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein.

Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli.

The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis. Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes. Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one. The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged. The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax. Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.

Drug Action of Ritodrine on the Sarcoplasmic-Reticulum Ca2+-ATPase from Skeletal Muscle

Ritodrine inhibits the steady-state Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles in a dose-dependent manner, The observed K0.5 value for inhibition (∼3 mM) gives proof of a low-affinity interaction. Ritodrine also interferes with the steady-state Ca2+ transport ability decreasing the maximal rate without modification of the Ca2+ or ATP affinity for the enzyme, This is consistent with an absence of competition for the transport and the catalytic sites. Analysis of the catalytic and transport cycle shows that: (i) ritodrine inhibits the phosphorylation partial reaction, This is supported by pre-steady-state kinetic experiments of Ca2+ transport and also by the temperature dependence of the phosphoenzyme level. (ii) At high ritodrine concentrations the dephosphorylation step becomes rate-limiting. This is suggested by the biphasic profile (V-shape) of phosphoenzyme accumulation at different ritodrine concentrations, This was also confirmed by chase experiments of radioactive phosphoenzyme decomposition at steady state. These data reveal a complex pattern of inhibition involving two sites for interaction with low and high ritodrine concentrations. It is envisaged that ritodrine does not exert any direct effect on the smooth muscle sarcoplasmic reticulum Ca2+-ATPase when used in the treatment of preterm birth.

Induction of αvβ3 integrin-mediated attachment to extracellular matrix in β1 integrin (CD29)-negative B cell lines

β 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the β 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the β 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the α v β 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the α v β 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ α v β 3, and these lines similarly lacked expression of β 1 integrin. These results indicate that α v β 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated α v β 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.

Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii

The effects of nitric oxide (NO) on the membrane-associated form of the nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii have been investigated. In the presence of H 2 and an electron acceptor (turnover conditions), NO acts as a noncompetitive inhibitor vs. methylene blue ( K i = 12 μM). There is no element of competition between NO and H 2, implying that the site of NO action is not the H 2-activating site of the hydrogenase. When the membrane-associated hydrogenase is incubated under non-turnover conditions, the enzyme is irreversibly inactivated by NO in a time-dependent process. The inactivation is a non-saturable, pseudo-first-order process which is consistent with a direct chemical reaction between NO and the hydrogenase. Kinetic evidence is presented which is compatible with an interaction between NO and a redox-active component other than the H 2-activating site on the enzyme. The complex inhibition pattern of NO has been interpreted in terms of two distinct interactions of NO with iron-sulfur centers of the hydrogenase.

Silver toxicity in a nitrogen-fixing Cyanobacterium

Exposure ofNostoc muscorum to combinations of Ag + Cr, Ag + Ni, Ag + PB and Ag, Cr, Ni and Cr alone brought about a complex pattern of inhibition of growth, NO3, NH4 and14CO2 uptake and nitrogenase activity. The sensitivity hierarchy of different parameters to metal combinations may be given as: (a) inhibition of NH4 uptake and nitrogenase was almost identical for Ag + Ni, Ag + Cr and Ag + Pb; (b) the order of inhibition of growth and NO3 uptake was Ag + Cr > Ag + Ni > Ag + Pb; (c) the combination of Ag + Pb was least toxic for growth, nitrogenase, NO3 and NH4 uptake, but most toxic for14CO2 uptake; (d) the toxicity hierarchy for all parameters together was14C02 > nitrogenase > NH4 uPtake > NO3 uptake > growth. Hence, carbon fixation may be employed for biological monitoring of metal toxicity in a laboratory microcosm and possibly in aquatic ecosystems.

Nature of taurodehydrocholic acid uptake in rat hepatocytes.

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

1. 1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine. 2. 2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5. 3. 3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site. 4. 4. The K I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane. 5. 5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by β- d-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10–60 m m), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 m m. In contrast, at high concentrations (80–320 m m), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 m m. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = −0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site ( K i = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower k cat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.

Kinetic mechanism in the direction of oxidative decarboxylation for NAD-malic enzyme from Ascaris suum.

Measurement of the initial rate of the malic enzyme reaction varying the concentration of NAD at several different fixed levels of Mg2+ (0.25-1.0 mM) and a single malate concentration gave a pattern which intersects to the left of the ordinate. Repetition of this initial velocity pattern at several additional malate concentrations and treatment in terms of a terreactant mechanism suggests an ordered mechanism in which NAD adds prior to Mg2+ which must add prior to malate. On the other hand, when a broader concentration range of Mg2+ (0.25-50 mM) is used, data are consistent with a random mechanism in which Mg2+ must add prior to malate. By use of product inhibition studies, pyruvate is competitive vs. malate and noncompetitive vs. NAD, while NADH is competitive vs. NAD and noncompetitive vs. malate. These results are consistent with the random addition of substrates and further suggest rapid equilibrium random release of products. Tartronate, a dead-end analogue of malate, is competitive vs. malate and noncompetitive vs. NAD. Thio-NAD is a slow substrate which is used at 2.4% the maximum rate of NAD. When used as a dead-end analogue of NAD, thio-NAD is competitive vs. NAD and gives a complex inhibition pattern vs. malate in which competitive inhibition is apparent at low concentrations of malate (less than 12.5 mM), and this changes to uncompetitive inhibition at high concentrations of malate (greater than 12.5 mM). These data are consistent with a steady-state random mechanism in the direction of oxidative decarboxylation in which Mg2+ adds in rapid equilibrium prior to malate.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [(3)H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.