Conditions Of Substrate Saturation Research Articles

Recombinant human growth hormone and insulin-like growth factor-1 do not affect mitochondrial derived highly reactive oxygen species production in peripheral blood mononuclear cells under conditions of substrate saturation in-vitro.

BackgroundThe purpose of this study was to investigate the mitochondrial effects exerted by physiological and supra-physiological concentrations of recombinant human growth hormone (rhGH) and recombinant insulin-like growth factor-1 (rIGF-1) under conditions of substrate saturation in peripheral blood mononuclear cells (PBMCs).MethodsPBMCs from healthy male subjects were treated with either rhGH, at concentrations of 0.5, 5 and 50 μg/L, or rIGF-1 at concentrations of 100, 300 and 500 μg/L for 4 h. Mitochondrial membrane potential (Δψm) and mitochondrial levels of highly reactive oxygen species (hROS) were subsequently analysed. This analysis was performed by flow cytometry in digitonin permeabilized cells, following treatment with saturating concentrations of various respiratory substrate combinations and the use of specific electron transport chain (ETC.) complex inhibitors, enabling control over both the sites of electron entry into the ETC. at complexes I and II and the entry of electrons from reduced carriers involved in β-oxidation at the level of ubiquinol.ResultsNeither rhGH nor rIGF-1 exerted any significant effect on Δψm or the rate of hROS production in either lymphocyte or monocyte sub-populations under any of the respiratory conditions analysed.ConclusionThat neither hormone was capable of attenuating levels of oxidative stress mediated via either complex I linked respiration or lipid-derived respiration could have serious health implications for the use of rhGH in healthy individuals, which is frequently associated with significant increases in the bioavailability of free fatty acids (FFA). Such elevated supplies of lipid-derived substrates to the mitochondria could lead to oxidative damage which would negatively impact mitochondrial function.

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation. Branched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2.

Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

Kinetic equivalence of the subunits of liver alcohol dehydrogenase.

1 The kinetics of two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single-turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic-suicide experiments. 2 Amplitudes of the slow phase 328–330-nm absorbance changes occurring during the enzymic reduction of various para-substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects. 3 Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330-nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined. 4 It has been concluded in recent publications that an ordered ternary-complex mechanism involving equivalent sites is insufficient to account for the transient-state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non-equivalence of the enzymic sites. Subunit interactions resulting in a “half-site7rdquo; reactivity of the enzyme are definitely not at hand.

Kinetic Properties of the Enzyme-Substrate System: A Basis for Immediate Temperature Compensation

One-minimum U-shaped temperature profiles of the dissociation constant ( K m ) have been observed experimentally with a variety of enzyme–substrate (E–S) systems. The increase of E–S affinity with falling temperature (“positive thermal modulation of affinity”), which opposes the cold-induced reduction in catalytic velocity, has been often interpreted as significant for both immediate and evolutionary temperature compensations and of major importance in setting thermal limits in ectothermic organisms. This role was denied to enzymes from endotherms, on the ground that their minimal K m values were situated well below their normal body temperature. Evidence is presented in this report that affinity changes described by U-shaped profiles can simply be the consequence of intrinsic kinetic properties of the E–S system. Theoretical modeling is achieved by combining the classical expression for the Michaelis constant with Transition State Theory expressions for the three rate constants involved. It provides for the U-shape of the K m vs. T profile and allows for the derivation of an equation for identifying its inversion point. Modeling of V max and V min (reaction velocity under conditions of substrate saturation and of dilution, K m ⪢[ S], respectively) is also included. An expression was formulated for predicting the “critical temperature,” T C, corresponding to the low-temperature break in Arrhenius lines. Using existing K m data from literature, concerning a variety of E–S systems, our modeling proved to be highly satisfactory. Our own experiments show that glucose uptake by rat brain synaptosomes can be regarded as a special case of basically the same kinetic scheme, and that the U-shaped temperature modulation of apparent K m for glucose conversion is also in full agreement with our kinetic modeling. These experiments indicate that positive thermal modulation, although based on intrinsic kinetic properties of the underlying E–S system, may have an adaptive role in endotherms as well, linked, however, to their tolerance to hypothermia.

Kinetics and mechanism of the hydrolysis of depsipeptides catalyzed by the beta-lactamase of Enterobacter cloacae P99.

The steady-state kinetics and mechanism of the hydrolysis and aminolysis of a series of acyclic depsipeptides, catalyzed by the class C beta-lactamase of Enterobacter cloacae P99, have been studied in order to more firmly establish the nature of the transition states involved. The class C beta-lactamase of Enterobacter cloacae P99 was employed. The depsipeptide substrates contained a constant acyl group, (phenylacetyl)glycyl, and chemically different leaving groups, m-carboxyphenoxide, m-carboxythiophenoxide, 3-carboxyl-4-nitrophenoxide, lactate, and thiolactate. Evaluation of the steady-state kinetic parameters and the effect of the alternative nucleophile methanol on these parameters and on the product distribution showed that deacylation was largely rate-determining to turnover of the aryl esters under conditions of substrate saturation, while acylation was rate-determining to the alkyl esters. The earlier conclusion [Govardhan & Pratt (1987) Biochemistry 26, 3385-3395] that acylation largely limited the turnover of the aryl esters was shown to be an artifact of phosphate buffer inhibition. The aminolysis of both the aryl the alkyl esters by D-phenylalanine was influenced by binding of the substrate at a second binding site on the acyl-enzyme intermediate. A study of inhibiton of the hydrolysis of (phenylacetyl)-glycyl-D-thiolactate by the aminolysis product (phenylacetyl)glycyl-D-phenylalanine indicated that the second binding site is also available for ligands to bind the free enzyme and to the noncovalent Michaelis complex with this substrate. It is likely that penicillin-recognizing enzymes in general, both beta-lactamases and DD-peptidases, possess an extended substrate-binding site into which a variety of small ligands may bind at any point along the reaction coordinate and, to a greater or lesser extent depending on circumstances, affect catalysis.

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states

Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.

Use of isotope effects to characterize intermediates in mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine.

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which bound alpha-aminoacetophenone is generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (Mangold, J.B., and Klinman, J.P. (1984) J. Biol. Chem. 259, 7772-7779). Based on the assumption that the ketone radical is the inhibitory intermediate, an analogous system was predicted and verified (Bossard, M.J., and Klinman, J.P. (1986) J. Biol. Chem. 261, 16421-16427). In the present study, the role of alpha-aminoacetophenone as the proposed intermediate in the inactivation by beta-chlorophenethylamine was examined in greater detail. From the interdependence of tyramine and alpha-aminoacetophenone concentrations, ketone inactivation is concluded to occur at the substrate site as opposed to potential binding at the reductant-binding site. Using beta-[2-1H]- and beta-[2-2H]chlorophenethylamine, the magnitude of the deuterium isotope effect on inactivation under second-order conditions has been found to be identical to that observed under catalytic turnover, D(kappa inact/Ki) = D(kappa cat/Km) = 6-7. By contrast, the isotope effect on inactivation under conditions of substrate and oxygen saturation, D kappa inact = 2, is 3-fold smaller than that seen on catalytic turnover, D kappa cat = 6. This reduced isotope effect for inactivation is attributed to a normal isotope effect on substrate hydroxylation followed by an inverse isotope effect on the partitioning of the enol of alpha-aminoacetophenone between oxidation to a radical cation versus protonation to regenerate ketone. These findings are unusual in that two isotopically sensitive steps are present in the inactivation pathway whereas only one is observable in turnover.

Open circuit relaxation behaviour of enzyme electrodes

The field of enzyme electrodes has attracted a great deal of interest in recent years [l-7] and quite a number of papers have appeared dealing with steady state enzyme redox catalysis with a soluble redox mediator in solution [l-6]. Most of these studies, however, involve amperometric techniques and measure the stationary current that generates a flux of mediator at the electrode surface [2]. Under conditions of substrate saturation the steady state kinetics of enzyme reoxidation by the redox mediator can be treated as an extension of the theory of voltammetry for a catalytic mechanism of the type:

Growth-rate inhibition of acetoclastic methanogens by ammonia and pH in poultry manure digestion

The maximum growth rate ( μ m ) of acetoclastic methanogens was measured at ammonia concentrations between 0·55 and 0·74 mol/liter (7·7 and 10·4 g ammonia-N/liter) and a pH between 7·80 and 7·93. The observed μ m varied between 0·0046 and 0·0000 h −1 under these experimental conditions. The μ m was calculated by monitoring the gas production rate of batch reactors under conditions of substrate saturation (maximum growth rate in prevailing environment); the acetate concentration during all experiments was above 0·025 mol/liter (1·5 g/liter). It was shown that the pH and total ammonia concentration are the predominant inhibition factors for acetoclastic methanogens under these conditions. The best mathematical description for the inhibition of the growth rate is given by an equation which contains the total ammonia concentration as well as the pH, and this description was significantly better than a description using a calculated free ammonia concentration. The results can be used to calculate a minimal hydraulic retention time for acetate degradation in poultry manure digesters.

Carbamoyl phosphate biosynthesis and partition in pyrimidine and arginine pathways of Escherichia coli. In situ properties of carbamoyl-phosphate synthase, ornithine transcarbamylase and aspartate transcarbamylase in permeabilized cells.

A procedure for the permeabilization of Escherichia coli cells was adapted to the in situ determination of the catalytic and regulatory properties of the enzymes responsible for the biosynthesis of carbamoyl phosphate and its utilization in the pyrimidine and arginine pathways. Differences in enzyme sensitivity to effectors and changes in pH dependence were observed. Partition of carbamoyl phosphate in the two metabolic pathways could be measured under conditions of substrate saturation. The results obtained will allow to test experimentally the theoretical predictions made by A. Goldbeter (1973) PhD thesis, Université Libre de Bruxelles, on the distribution of carbamoyl phosphate and the oscillation of its intracellular concentration.

Kinetics and mechanism of the serine .beta.-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.

CO2 is the inorganic carbon substrate of NADP malic enzymes from Zea mays and from wheat germ.

NADP malic enzyme (EC 1.1.1.40) was extracted and partially purified from the green leaves of Zea mays var. Felix and from wheat germ. The active inorganic carbon species for both enzymes was, in contrast to an earlier report, CO2 not HCO3-. The apparent Km, CO2 for the maize enzyme was 1.2 mM and the apparent Km, CO2 for the wheat germ preparation was 4.2 mM under conditions of substrate saturation, pH 7.3 and 17 degrees C. These observations support the views that CO2, rather than HCO3- as has been suggested, is produced in bundle-sheath chloroplasts and that the reaction mechanism catalysed by plant cytosolic and chloroplastic NADP malic enzymes is similar to that proposed for avian NADP malic enzymes.

13C and 15N uptake by marine phytoplankton—IV. Uptake ratios and the contribution of nitrate to the productivity of Antarctic waters (Indian Ocean sector)

Significant latitudinal gradients in inorganic carbon and nitrate uptake were observed in the Indian sector of the Antarctic Ocean. Carbon uptake weas directly related to photosynthetically available radiation (PAR), while nitrate uptake was inversely related to PAR. This led to a direct relationship between carbon:nitrate uptake ratios and PAR both within and between stations. Field measurements as well as laboratory experiments on cultures of two diatoms isolated from the study area show that ammonium uptake decreases with time after addition of ammonium, under conditions of substrate saturation. This phenomenon, as well as other problems associated with measurements of ammonium uptake, makes it difficult to estimate the contribution of this compound to primary production from direct uptake measurements. More reliable estimates can be obtained from values of carbon:nitrate uptake ratios determined from double labeling experiments. These ratios are lower than the Redfield ratio south of the polar front, and indicate that nitrate can account for most of the primary production in offshore areas during the austral summer.

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum.

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).

Role of conjugation and red blood cells for inactivation of circulating normetanephrine.

Plasma and red blood cell (RBC) concentrations of normetanephrine (NMN), in both free and glucuronide-conjugated forms, were measured before, during, and after forced immobilization, an intense stressor of the sympathoadrenal system of rats. In this study NMN glucuronide was deconjugated by enzymatic hydrolysis; free and total NMN were assayed by radioenzymatic, thin-layer chromatographic procedures. In plasma, free NMN and NMN glucuronide are 777 +/- 99 and 792 +/- 74 pg/ml, respectively, when rats are at rest. Both free NMN and NMN glucuronide increased about 200% after 15 min of stress; in absolute amounts, increases were equivalent to that of the simultaneous increase in norepinephrine (NE). At 2 h of stress, NMN glucuronide, but not free NMN, increased further and significantly. The mean concentration of RBC-free NMN is about 50 times higher than that of plasma-free NMN, and it did not change significantly during stress; RBCs do not contain conjugated NMN. RBC NMN levels showed a strong correlation with RBC catechol methyltransferase activity. The latter seems to operate under conditions of substrate saturation; an acute release of NE leads to temporary storage of NE in RBCs but not conversion to NMN. The results indicate that conjugation of NMN with glucuronic acid is an important route for inactivation of plasma NMN formed during forced immobilization stress, whereas free NMN does not accumulate in RBCs during stress.

Phosphorylation of rhodopsin and quenching of cyclic GMP phosphodiesterase activation by ATP at weak bleaches.

Light and GTP-dependent cyclic GMP phosphodiesterase activation of rod disk membranes is rapidly quenched by ATP. Maximum speed of this effect occurs only with the weakest bleaches. Though it has been proposed that ATP mediates its effect through rapid phosphorylation of bleached rhodopsin, previous workers have found phosphorylation kinetics too slow by more than an order of magnitude to be causal in quenching of cyclic GMP phosphodiesterase activation. In this report, we use preparations retaining more endogenous rhodopsin kinase, higher specific activity ATP, and cyclic GMP phosphodiesterase quenching conditions to show that ATP-dependent multiple phosphorylation of rhodopsin at very weak bleaches (10(-5)) is complete in less than 2 s, easily compatible with cyclic GMP phosphodiesterase quench times of 4 s measured under identical conditions. Thus, it seems likely that previous efforts to achieve high 32P counts by using large bleaches have produced conditions of substrate saturation where much longer times to completion are caused by a very large ratio of substrate to enzyme velocity. Such conditions are not appropriately compared to those that support rapid quenching. We conclude that the speed of rhodopsin phosphorylation is, in fact, adequate to explain ATP quenching of cyclic GMP phosphodiesterase activation.

Transient situations in nitrate assimilation by marine diatoms. 4. Non-linear phenomena and the estimation of the maximum uptake rate

Under conditions of substrate saturation and otherwise constant environmental parameters, the uptake rate (V'max) of nitrate (and other nitrogen compounds) can vary widely following the addition of nitrate (or other nitrogen compounds) to medium containing unicellular algae under various physiological states. Cases are presented in which V'max changes in different directions during the incubation, over a time scale shorter than the generation time or the day-night periodicity. The influence of each pattern is discussed in relation to the Michaelis-Menten equation. Examples are given in which the experimental protocol will yield data reflecting a relationship between uptake rate and time rather than uptake rate and substrate concentration. It is shown that the two methods now in use for estimating V'max will not give similar results unless the uptake rate remains constant over the duration of the measurement. In nature as well as in culture, this condition seems to be seldom met. For short-term incubations, physical phenomena such as adsorption and diffusion are difficult to separate from biological ones. As the incubation duration increases, biological processes such as enzyme induction, feedback regulation and product excretion will lead to deformations of classical enzymatic kinetics.

Reduced Hexose Transport by Enterocytes Associated with Rapid, Noninjurious Rejection of Trichinella spiralis from Immune Rats

Rats immunized against Trichinella spiralis rejected larvae from a challenge infection within minutes. Infectivity of these once-rejected larvae for nonimmune rats following intestine-to-intestine transfer, infectivity for nonimmune mice following oral inoculation, and normal development in these mice indicated that the parasite did not sustain immediate or long term damage as a result of the host's immune response. Associated with rapid worm rejection was a change in host intestinal function - altered absorption. Uptake of a hexose, 14C-beta-methyl-D-glucoside, was studied in intestinal segments isolated from rats before and 30 min after primary and secondary infections. Larvae were administered intraintestinally. Absorption was measured using a tissue accumulation method, under conditions of substrate saturation (30 mMolar) and over a 5-min period. 3H-mannitol was used as a marker to measure extracellular water, hence, extracellular sugar. Following challenge infection of previously infected (immunized) hosts, there was a significant reduction (22%; P less than 0.05) in hexose absorption as compared to the uptake rate before challenge. In contrast, cellular uptake before and 30 min after primary infection was similar. These findings are presented in support of the hypothesis that the failure of T. spiralis larvae to infect immune hosts is the result, not of direct worm damage, but of functional changes in gut epithelium, the habitat of the parasite.

Angiotensin-converting enzyme: Serum levels during normal pregnancy

ACE activity of the serum of 52 normal pregnant women was measured in vitro under conditions of substrate saturation with Hip-His-Leu as substrate. The product His-Leu was measured by fluorimetry after reaction with o-phthaldehyde. ACE activity (nmol/min/ml serum) was 30.6 +/- 7.8, 28.8 +/- 7.4, and 30.9 +/- 8.2 for the first, second, and third trimester of pregnancy, respectively. No statistically significant differences (p greater than 0.05) in ACE activity were detected among the three trimesters of normal pregnancy with either serum volume or serum protein as reference value. These values are within the range reported by Friedland and Silverstein13 for 51 male and seven female healthy blood bank donors. We conclude that the evolution of normal pregnancy does not significantly modify the levels of ACE in peripheral blood serum.