Assessment of methods for improving tracer estimation of non-steady-state rate of appearance.

The most common approach for estimating substrate rate of appearance (R(a)) is use of the single-pool model first proposed by R. W. Steele, J. S. Wall, R. C. DeBodo, and N. Altszuler. (Am. J. Physiol. 187: 15-24, 1956). To overcome the model error during highly non-steady-state conditions due to the assumption of a constant volume of distribution (V), two strategies have been proposed: 1) use of a variable tracer infusion rate to minimize tracer-to-tracee ratio (TTR) variations (fixed-volume approach) or 2) use of two tracers of the same substrate with one infused at a constant rate and the other at a variable rate (variable-volume approach or approach of T. Issekutz, R. Issekutz, and D. Elahi. Can. J. Physiol. Pharmacol. 52: 215-224, 1974). The goal of this study was to compare the results of these two strategies for the analysis of the kinetics of glycerol and glucose under the non-steady-state condition created by a constant infusion of epinephrine (50 ng. kg(-1). min(-1)) with the traditional approach of Steele et al., which uses a constant infusion and fixed volume. The results showed that for glucose and glycerol the estimates of R(a) obtained with the constant and the variable tracer infusion rate and the equation of Steele et al. were comparable. The variable tracer infusion approach was less sensitive to the choice of V in estimating R(a) for glycerol and glucose, although the advantage of changing the tracer infusion rate was greater for glucose than for glycerol. The model of Issekutz et al. showed instability when the ratio TTR(1)/TTR(2) approaches a constant value, and the model is more sensitive to measurement error than the constant-volume model for glucose and glycerol. We conclude that the one-tracer constant-infusion technique is sufficient in most cases for glycerol, whereas the one-tracer variable-infusion technique is preferable for glucose. Reasonable values for glucose R(a) can be obtained with the constant-infusion technique if V = 145 ml/kg.     

Reutilization of DNA catabolites in granulocytopoiesis.

Different DNA labelling procedures for the proliferating granulocyte precursor compartments of rat bone marrow were evaluated by studying the appearance of labelled granulocytes in the blood stream by means of autoradiography. 3-H-thymidine was administered by single injection and continuous infusion. 125-I-Iododeoxyuridine, a DNA precursor showing a neglegible extent of reutilization, was studied in comparison. Labelling patterns of blood neurtrophils were identical after single injection and continuous infusion of 3-H-thymidine, while a different pattern was observed after use of a single injection of 125-I-Iododeoxyuridine. The results provide evidence that reutilization of label is an important event to be considered when kinetics of granulocytopoiesis are studied after application of 3-H-thymidine and indicate that reutilization occurs at the level of thymidinemonophosphate in this cell system.     

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.     

Effect of infusion of "tris-galactosyl-cholesterol" on plasma cholesterol, clearance of lipoprotein cholesteryl esters, and biliary secretion in the rat

As shown by us previously (van Berkel et al. 1985. J. Biol. Chem. 260: 2694-2699 and van Berkel et al. 1985. J. Biol. Chem. 260: 12203-12207) the clearance of both low density lipoproteins (LDL) and high density lipoproteins (HDL) from the blood can be greatly enhanced by pretreatment of these lipoproteins with a tris-galactosylated cholesterol derivative, which makes these particles recognizable by hepatic galactosyl-receptors. Here we report that intravenous infusion of the (water-soluble) tris-galactosyl-cholesterol in rats caused a dose-dependent decrease of the plasma cholesterol level. This fall was sustained long after termination of the infusion. It was not observed upon infusion of tris-glucosyl-cholesterol. The fall in plasma cholesterol was accompanied by an increase in hepatic cholesterol. Upon injection of rat HDL and LDL labeled in their cholesteryl ester moieties, plasma clearance of label in both lipoproteins was enhanced in rats infused with tris-galactosyl-cholesterol, the stimulation being more pronounced when the label was in HDL. The appearance of label in bile was also enhanced in the rats receiving the compound, again more markedly when the label was given as HDL. Ninety four percent or more of the radioactivity excreted in the bile was in the form of bile salts, with conjugated cholate being the major species in both control and treated rats; 6% or less of the radioactivity in the bile was as free cholesterol. Infusion of tris-galactosyl-cholesterol constitutes a new and defined method of lowering plasma lipoprotein levels by enhancing their uptake in the liver.     

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.     

Estimating multilevel logistic regression models when the number of clusters is low: a comparison of different statistical software procedures

Multilevel logistic regression models are increasingly being used to analyze clustered data in medical, public health, epidemiological, and educational research. Procedures for estimating the parameters of such models are available in many statistical software packages. There is currently little evidence on the minimum number of clusters necessary to reliably fit multilevel regression models. We conducted a Monte Carlo study to compare the performance of different statistical software procedures for estimating multilevel logistic regression models when the number of clusters was low. We examined procedures available in BUGS, HLM, R, SAS, and Stata. We found that there were qualitative differences in the performance of different software procedures for estimating multilevel logistic models when the number of clusters was low. Among the likelihood-based procedures, estimation methods based on adaptive Gauss-Hermite approximations to the likelihood (glmer in R and xtlogit in Stata) or adaptive Gaussian quadrature (Proc NLMIXED in SAS) tended to have superior performance for estimating variance components when the number of clusters was small, compared to software procedures based on penalized quasi-likelihood. However, only Bayesian estimation with BUGS allowed for accurate estimation of variance components when there were fewer than 10 clusters. For all statistical software procedures, estimation of variance components tended to be poor when there were only five subjects per cluster, regardless of the number of clusters.     

Effect of lactate and beta-hydroxybutyrate infusions on brain metabolism in the fetal sheep

Brain uptake of substrates other than glucose has been demonstrated in neonatal but not fetal animals in vivo. This study was undertaken to investigate the ability of the fetal sheep brain to use potential alternative substrates when they were provided in increased amounts. Brain substrate uptake was measured in chronically catheterised fetal sheep during 2-h infusions of neutralised lactate (n = 12) or beta-hydroxybutyrate (n = 12). Despite large increases in fetal arterial lactate and beta-hydroxybutyrate during the respective infusions, no significant uptake of either substrate was demonstrated. However during both types of infusion, the brain arterio-venous difference for glucose decreased 30% (P less than 0.05). Since the brain arterio-venous difference for oxygen was unchanged, and blood flow to the cerebral hemispheres (measured in 11 studies) was also unchanged, the infusions appeared to cause a true decrease in brain glucose uptake. This decrease paralleled the rise in lactate concentration during lactate infusions, and the rise in lactate and butyrate concentrations during the butyrate infusions. Both substrates have metabolic actions that may inhibit brain glucose uptake. We speculate that the deleterious effects of high lactate and ketone states in the perinatal period may in part be due to inhibition of brain glucose uptake.     

Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes

An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.     

Time-dependent elimination of substrates flowing through the liver or kidney

Established steady-state models of elimination of flowing substrates by Michaelis-Menten kinetics in the intact liver and kidney are extended to time-dependent situations. It is shown how time-dependent distributions of substrate concentration can be calculated using steady-state results and a knowledge of the motion of fluid through the organs. The result is simplest when time-dependence is due to changes in substrate concentrations at the inlet, for example following injection or infusion. The case of the liver is treated in greater detail, and includes an evaluation of the instantaneous overall elimination rate.     

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.     

Anabolic effects of insulin and IGF-I in the ovine fetus are reduced by prolonged maternal fasting

Fetal nutritional stress may result in intrauterine growth restriction and postnatal insulin resistance. To determine whether insulin resistance can begin in utero, we subjected late-gestation (130-135 days) ewes to 120 h of complete fasting and compared the results with our previous work in fed ewes (38). We determined the effect of insulin and/or recombinant human (rh)IGF-I infusion on ovine fetal phenylalanine kinetics, protein synthesis, and phenylalanine accretion. Experimental infusates were 1) saline, 2) rhIGF-I plus a replacement dose of insulin (40 nmol IGF-I/h + 16 mIU insulin/h), 3) insulin (890 mIU/h), and 4) IGF-I plus insulin (40 nmol IGF-I/h + 890 mIU insulin/h). During hormone infusion, both glucose and amino acid concentrations were clamped at basal concentrations. Amino acid infusion was required during infusion of either hormone to maintain plasma concentrations constant. However, the amount required during insulin infusion was less than during IGF-I infusion and 40% less than the amount required during identical studies in nonfasted animals. Phenylalanine used for protein synthesis and accretion was increased compared with control animals but again less so than in the nonfasted animals. In contrast to nonfasted animals, neither hormone increased the fractional synthetic rate of skeletal muscle protein nor that of plasma albumin. These results indicate that a short but severe nutritional stress can significantly alter the fetal anabolic response to insulin even when both glucose and amino acid substrate supplies are restored. Therefore, adaptive responses characterized by insulin resistance begin in utero when the fetus is subjected to sufficient nutritional stress.     

Gastrointestinal tract, hepatic, hindlimb, and renal recovery of CO(2) in vivo.

Whole body oxidative rates of labeled substrates are often measured by collecting expired air and determining the amount of labeled CO(2) that is produced. However, the CO(2) produced may not be completely recovered under all circumstances, and there is a wide variation in values reported under different experimental conditions ( approximately 50-100%). The potential contribution of specific organs to this variation has not been defined. In vivo studies using healthy, postabsorptive, multicatheterized conscious canines were conducted to determine gastrointestinal tract, hepatic, hindlimb, and renal recoveries of NaH(14)CO(3) during a 180-min constant infusion [0.022+/-0.002 (SE) microCi x kg(-1) x min(-1)]. Before the constant infusion period, a bolus infusion of NaH(14)CO(3) (1.76+/-0.16 microCi/kg) was given, and the rate of decay in blood was measured over a 15-min period to determine pool size. The pool size for the distribution of (14)CO(2) was approximately 80% of the total body pool (16.0+/-1.7 liters). Whole body recovery was 97.2+/-6.7%. The recoveries across the liver, gut, leg, and kidney were 99.9+/-1.3, 98.0 +/-1.4, 96.7+/-2.6, and 99.9+/-2.1%, respectively. In conclusion, hepatic, gastrointestinal tract, hindlimb, and renal recoveries of CO(2) in vivo were near 100%, suggesting that CO(2) loss is not greater in gluconeogenic organs and that corrections for incomplete recovery of CO(2), when measuring oxidation of substrates across these organs under normal postabsorptive conditions, would be very minor.     

The use of isotope effects to determine enzyme mechanisms

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/K, and the ratio of the vertical intercepts is the isotope effect on V(max). This is the only way to determine V(max) isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/K of the labeled reactant. This is the method used for tritium or (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/K is given by: (x)(V/K)=((x)k+c(f)+c(r)(x)K(eq))/(1+c(f)+c(r)) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, (13)C, (15)N, or (18)O), and (x)(V/K), (x)k, and (x)K(eq) are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c(f) and c(r) are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c(f), or the first product released or the one involved in the perturbation for c(r). The intrinsic isotope effect, (x)k, can be estimated by comparing deuterium and tritium isotope effects on V/K, or by comparing the deuterium isotope effect with (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.     

Taurine kinetics assessed using [1,2-13C2]taurine in healthy adult humans

To assess the dynamics of taurine metabolism in vivo, two sets of studies were carried out in healthy volunteers. First, pilot studies were carried in a single human subject to determine the time course of plasma and whole blood isotope enrichment over the course of an 8-h, unprimed continuous infusion of [1,2-(13)C(2)]taurine. Second, five healthy adult males received two tracer infusions on separate days and in randomized order: 1) a 6-h continuous infusion of [1,2-(13)C(2)]taurine (3.1 +/- 0.2 micromol x kg(-1) x h(-1)) and 2) a bolus injection of [(13)C(2)]taurine (3.0 +/- 0.1 micromol/kg). Isotope enrichments in plasma and whole blood taurine were determined by gas chromatography-mass spectrometry. The pilot experiments allowed us to establish that steady-state isotope enrichment was reached in plasma and whole blood by the 5th h of tracer infusion. The plateau enrichment reached in whole blood was lower than that obtained in plasma taurine (P < 0.02). In the second set of studies, the appearance rate (R(a)) of plasma taurine, determined from continuous infusion studies was 31.8 +/- 3.1 micromol x kg(-1) x h(-1). After a bolus injection of tracer, the enrichment decay over the subsequent 2 h was best fitted by a two-exponential curve. Taurine R(a) was approximately 85% higher when determined using the bolus injection technique compared with continuous infusion of tracer. We conclude that 1) taurine R(a) into plasma is very low in healthy postabsorptive humans, and, due to taurine compartmentation between the extra- and intracellular milieus, may represent only interorgan taurine transfer and merely a small fraction of whole body taurine turnover; and 2) the bolus injection technique may overestimate taurine appearance into plasma. Further studies are warranted to determine whether alterations in bile taurine dynamics affect taurine R(a).     

Radioactive labeling of a natural assemblage of marine sedimentary bacteria and microalgae for trophic studies: An autoradiographic study

Autoradiography was used to examine critical questions for trophic studies concerning the uptake of radioactive tracers by a natural assemblage of sedimentary microorganisms. Labeled organic substrates ([³H]-acetate and [³H]-thymidine) were taken up only by heterotrophic bacteria, and [¹⁴C]-bicarbonate was taken up only by microalgae. Only approximately 2% of the bacterial assemblage took up detectable quantities of either [³H]-acetate or [³H]-thymidine, regardless of whether labeled substrates were delivered to sediments via slurries or by injection with a microliter syringe. Significantly more diatoms were labeled when [¹⁴C]-bicarbonate was delivered to sediments by the injection method (75%) as compared to the slurry method (50%). These results indicate that radio-active tracers can be used in natural sediments to selectively label potential microbial food of invertebrate grazers. Only a small proportion of bacteria, however, may actually use a labeled substrate, which introduces a large uncertainty into the conversion of radioactivity in grazers to the number of bacteria consumed. Finally, the use of disruptive methods (e.g., slurries) to deliver labels to sediments does not increase the proportion of microorganisms that become labeled. Thus, given the variety of artifacts that may be associated with the use of sediment slurries, it is probably advisable to use nondisruptive methods to deliver substrates to sediments.     

Variables of myocardial backdiffusion,determined with 17-IODO-131 heptadecanoic acid in the normal dog heart

Summary Under normal and ischemic conditions backdiffusion of radiolabeled non-esterified fatty acids (NEFA) has been demonstrated. In the fasted normal canine heart the extraction fraction (EF) during interventions with glucose or lactate loading, vasodilation, and metabolic level augmentation was determined, and compared with the control EF. Backdiffusion alterations were deduced from the EF changes. After iv injection of 17-iodo-131 heptadecanoic acid (IHDA), 11 blood samples were drawn from aorta and coronary sinus in a time period of 60 minutes. In the control and vasodilation group the EF slowly decreased from 40 to 10%. In contrast, the EF in the noradrenaline group was constant. During glucose and lactate infusion the EF became negative within 10 min and remained negative. These results suggest that during physiological circumstances backdiffusion is determined by the metabolic level of the heart and its substrate availability.     

Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: implications for the reaction mechanism

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pd) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of Pi, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Brain Arachidonic Acid Incorporation and Precursor Pool Specific Activity During Intravenous Infusion of Unesterified [3H]Arachidonate in the Anesthetized Rat

Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [³H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly ( t _1/2 < 1 min) to steady-state values that averaged <5% of plasma specific activity. Incorporation was rapid, as >85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.     

Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles

A detailed kinetic analysis of purified yeast membrane-associated phosphatidate phosphatase was performed using Triton X-100/phosphatidate mixed micelles. Enzyme activity was dependent on the bulk and surface concentrations of phosphatidate. These results were consistent with the "surface dilution" kinetic scheme (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) where phosphatidate phosphatase binds to the mixed micelle surface before binding to its substrate and catalysis occurs. Phosphatidate phosphatase was shown to physically associate with Triton X-100 micelles in the absence of phosphatidate, however, the enzyme was more tightly associated with micelles when its substrate was present. The enzyme had 5- to 6-fold greater affinity (reflected in the dissociation constant nKsA/chi) for Triton X-100 micelles containing dioleoyl-phosphatidate and dipalmitoyl-phosphatidate when compared to micelles containing dicaproyl-phosphatidate. The Vmax for dioleoyl-phosphatidate was 3.8-fold higher than the Vmax for dipalmitoyl-phosphatidate, whereas the interfacial Michaelis constant chi KmB for dipalmitoyl-phosphatidate was 3-fold lower than the chi KmB for dioleoyl-phosphatidate. The specificity constants (Vmax/chi KmB) of both substrates were similar which indicated that dioleoyl-phosphatidate and dipalmitoyl-phosphatidate were equally good substrates. Based on catalytic constants (Vmax and chi KmB), dicaproyl-phosphatidate was the best substrate with an 11- and 14-fold greater specificity constant when compared to dioleoyl-phosphatidate and dipalmitoyl-phosphatidate, respectively.     

Relative efficiencies of the maximum-likelihood, neighbor-joining, and maximum-parsimony methods when substitution rate varies with site

The relative efficiencies of the maximum-likelihood (ML), neighbor- joining (NJ), and maximum-parsimony (MP) methods in obtaining the correct topology and in estimating the branch lengths for the case of four DNA sequences were studied by computer simulation, under the assumption either that there is variation in substitution rate among different nucleotide sites or that there is no variation. For the NJ method, several different distance measures (Jukes-Cantor, Kimura two- parameter, and gamma distances) were used, whereas for the ML method three different transition/transversion ratios (R) were used. For the MP method, both the standard unweighted parsimony and the dynamically weighted parsimony methods were used. The results obtained are as follows: (1) When the R value is high, dynamically weighted parsimony is more efficient than unweighted parsimony in obtaining the correct topology. (2) However, both weighted and unweighted parsimony methods are generally less efficient than the NJ and ML methods even in the case where the MP method gives a consistent tree. (3) When all the assumptions of the ML method are satisfied, this method is slightly more efficient than the NJ method. However, when the assumptions are not satisfied, the NJ method with gamma distances is slightly better in obtaining the correct topology than is the ML method. In general, the two methods show more or less the same performance. The NJ method may give a correct topology even when the distance measures used are not unbiased estimators of nucleotide substitutions. (4) Branch length estimates of a tree with the correct topology are affected more easily than topology by violation of the assumptions of the mathematical model used, for both the ML and the NJ methods. Under certain conditions, branch lengths are seriously overestimated or underestimated. The MP method often gives serious underestimates for certain branches. (5) Distance measures that generate the correct topology, with high probability, do not necessarily give good estimates of branch lengths. (6) The likelihood-ratio test and the confidence-limit test, in Felsenstein's DNAML, for examining the statistical of branch length estimates are quite sensitive to violation of the assumptions and are generally too liberal to be used for actual data. Rzhetsky and Nei's branch length test is less sensitive to violation of the assumptions than is Felsenstein's test. (7) When the extent of sequence divergence is < or = 5% and when > or = 1,000 nucleotides are used, all three methods show essentially the same efficiency in obtaining the correct topology and in estimating branch lengths.(ABSTRACT TRUNCATED AT 400 WORDS)