Quasi-integrals and the origin of bursting pH oscillations in an enzyme model system

Bursting Oscillation, Mixed-Mode oscillation, Slow Manifold, Quasi-Integral, slow-fast analysis, QSSA

A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

Sustained 24-hour blockade of the renin-angiotensin system: a high dose of a long-acting blocker is as effective as a lower dose combined with an angiotensin-converting enzyme inhibitor.

Whether a higher dose of a long-acting angiotensin II receptor blocker (ARB) can provide as much blockade of the renin-angiotensin system over a 24-hour period as the combination of an angiotensin-converting enzyme inhibitor and a lower dose of ARB has not been formally demonstrated so far. In this randomized double-blind study we investigated renin-angiotensin system blockade obtained with 3 doses of olmesartan medoxomil (20, 40, and 80 mg every day) in 30 normal subjects and compared it with that obtained with lisinopril alone (20 mg every day) or combined with olmesartan medoxomil (20 or 40 mg). Each subject received 2 dose regimens for 1 week according to a crossover design with a 1-week washout period between doses. The primary endpoint was the degree of blockade of the systolic blood pressure response to angiotensin I 24 hours after the last dose after 1 week of administration. At trough, the systolic blood pressure response to exogenous angiotensin I was 58% +/- 19% with 20 mg lisinopril (mean +/- SD), 58% +/- 11% with 20 mg olmesartan medoxomil, 62% +/- 16% with 40 mg olmesartan medoxomil, and 76% +/- 12% with the highest dose of olmesartan medoxomil (80 mg) (P = .016 versus 20 mg lisinopril and P = .0015 versus 20 mg olmesartan medoxomil). With the combinations, blockade was 80% +/- 22% with 20 mg lisinopril plus 20 mg olmesartan medoxomil and 83% +/- 9% with 20 mg lisinopril plus 40 mg olmesartan medoxomil (P = .3 versus 80 mg olmesartan medoxomil alone). These data demonstrate that a higher dose of the long-acting ARB olmesartan medoxomil can produce an almost complete 24-hour blockade of the blood pressure response to exogenous angiotensin in normal subjects. Hence, a higher dose of a long-acting ARB is as effective as a lower dose of the same compound combined with an angiotensin-converting enzyme inhibitor in terms of blockade of the vascular effects of angiotensin.

Effect on blood pressure and the renin-angiotensin system of repeated doses of the converting enzyme inhibitor CGS 14824A.

A new, orally active angiotensin converting enzyme (ACE) inhibitor, CGS 14824A, was evaluated in 12 healthy male volunteers. Two groups each of 6 volunteers were given 5 or 10 mg once daily p.o. for 8 days. Four hours after the first and the last morning doses, plasma angiotensin II, aldosterone and plasma converting enzyme activity had fallen, while blood angiotensin I and plasma renin activity had risen. Throughout the study, more than 90% inhibition of ACE was found immediately before giving either the 5 or 10 mg dose and 50% blockade was still present 72 h following the last dose. Based on the determination of ACE, there was no evidence of drug accumulation. No significant change in blood pressure or heart rate was observed during the course of the study. CGS 14824A was an effective, orally active, long-lasting and well tolerated converting enzyme inhibitor.

Involvement of the kallikrein-kinin system in the antihypertensive effect of the angiotensin converting enzyme inhibitors.

1. Studies were performed in normal subjects and in rats to assess the effect of angiotensin converting enzyme (ACE) inhibition on the kallikrein-kinin system. As ACE is identical to kininase II, one of the enzymes physiologically involved in bradykinin degradation, bradykinin may be expected to accumulate during ACE inhibition. 2. A competitive antagonist of bradykinin was used to explore in unanaesthetized rats the contribution of circulating bradykinin to blood pressure control under ACE inhibition. 3. No evidence was found for a role of this vasodilating peptide in the blood pressure lowering effect of acute ACE inhibition. 4. The plasma activity of carboxypeptidase N (= kininase I), another pathway of bradykinin degradation, remained intact during a 1 week course of treatment with an ACE inhibitor in normal subjects. This therefore indicates that bradykinin formed during ACE inhibition can still be metabolized.

Sustained 24-hour blockade of the renin-angiotensin system: a high dose of a long-acting blocker is as effective as a lower dose combined with an angiotensin-converting enzyme inhibitor

Résumé en français Jusqu'alors, il n'avait jamais été formellement démontré qu'une forte dose d'un antagoniste de l'angiotensine II à longue durée d'action pouvait être aussi efficace sur le blocage du système rénine-angiotensine que l'association d'un inhibiteur de l'enzyme de conversion avec le même antagoniste de l'angiotensine II à des doses plus faibles. Dans cette étude randomisée en double aveugle, nous avons étudié le blocage du système rénine-angiotensine obtenu avec trois doses d'olmesartan medoxomil (20, 40 et 80 mg) chez 30 volontaires sains que nous avons comparé au blocage obtenu par du lisinopril (20 mg), seul ou associé à de l'olmesartan medoxomil (20 et 40 mg). L'étude s'est déroulée en deux phases selon un design par crossover. A deux reprises, chaque volontaire à reçu durant une semaine l'un des six traitements possibles. Un intervalle d'une semaine a été respecté entre les deux phases (période de washout). L'objectif principal était d'étudier, 24 heures après la dernière dose, le blocage de l'élévation de la pression systolique en réponse à l'administration d'angiotensine I. Ce blocage était de 58% ± 19% (moyenne ± déviation standard) avec 20 mg de lisinopril, de 58% ± 11% avec 20 mg d'olmesartan medoxomil, de 62% ± 16% avec 40 mg d'olmesartan medoxomil, et de 76% ± 12% avec la plus forte dose d'olmesartan medoxomil (80 mg) (P=.016 versus 20 mg de lisinopril et P=.0015 versus 20 mg d'olmesartan medoxomil). Le blocage était de 80% ± 22% avec 20 mg de lisinopril associé à 20 mg d'olmesartan medoxomil et de 83% ± 9% avec 20 mg de lisinopril associé à 40 mg d'olmesartan medoxomil (P= .3 versus 80 mg d'olmesartan medoxomil). Ces résultats montrent, que chez les volontaires sains, une dose suffisamment élevée d'olmesartan medoxomil peut induire un blocage à 24 heures quasi complet de l'élévation de la pression artérielle en réponse à l'administration d'angiotensine I. De même, en terme de blocage de l'effet vasculaire de l'angiotensine I, une dose suffisamment élevée d'un antagoniste de l'angiotensine II de longue durée d'action est tout aussi efficace que ce même antagoniste à des doses plus faibles associé avec à un inhibiteur de l'enzyme de conversion.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml) or simple sugars (320-350 µg/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

Determination of Oxalate in Grass by FIA with a Spectrophotometric Detection System Using a Two Enzyme Reactor

A new methodology for soluble oxalic acid determination in grass samples was developed using a two enzyme reactor in an FIA system. The reactor consisted of 3 U of oxalate oxidase and 100 U of peroxidase immobilized on Sorghum vulgare seeds activated with glutaraldehyde. The carbon dioxide was monitored spectrophotometrically, after reacting with an acid-base indicator (Bromocresol Purple) after it permeated through a PTFE membrane. A linear response range was observed between 0.25 and 1.00mmol l-1 of oxalic acid; the data was fit by the equation A=-0.8(±1.5)+ 57.2(±2.5)[oxalate], with a correlation coefficient of 0.9971 and a relative standard deviation of 2% for n=5. The variance for a 0.25 mmol l-1 oxalic acid standard solution was lower than 4% for 11 measurements. The FIA system allows analysis of 20 samples per hour without prior treatment. The proposed method showed a good correlation with that of the Sigma Kit.

Oxalate determination in urine using an immobilized enzyme on Sorghum vulgare seeds in a flow injection conductimetric system

A flow-injection (FI) method was developed for the determination of oxalate in urine. It was based on the use of oxalate oxidase (E.C. 1.2.3.4) immobilized on ground seeds of the BR-303 Sorghum vulgare variety. A reactor was filled with this activated material, and the samples (200 μL) containing oxalate were passed through it, carried by a deionized water flow. The carbon dioxide produced by the enzyme reaction permeated through a microporous PTFE membrane, and was received in a water acceptor stream, promoting conductivity changes proportional to the oxalate concentration in the sample. The results obtained showed a useful linear range from 0.05 to 0.50 mmol dm-3. The proposed method, when compared with the Sigma enzymatic procedure, showed good correlation (Y = 0.006(±0.016) + 0.98(±0.019)X; r = 0.9995, Y = conductivity in μS, and X = concentration in mmol dm-3), selectivity, and sensitivity. The new immobilization approach promotes greater stability, allowing oxalate determination for 6 months. About 13 determinations can be performed per hour. The precision of the proposed method is about ± 3.2 % (r.s.d).

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

The N-terminal domain of Escherichia coli enzyme I of the phosphoenolpyruvate/glycose phosphotransferase system: molecular cloning and characterization.

The bacterial phosphoenolpyruvate/glycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to sugars concomitant with their translocation. The first two steps of the phosphotransfer sequence are PEP <--> Enzyme I (EI) <--> HPr (the histidine-containing phosphocarrier protein). We have proposed that many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition. EI monomer (63.5 kDa) comprises two major domains: a flexible C-terminal domain (EI-C) and a protease-resistant, structurally stable N-terminal domain (EI-N) containing the active site His. Trypsin treatment of Salmonella typhimurium EI yielded EI-N, designated EI-N(t). Homogeneous recombinant Escherichia coli EI-N [i.e., EI-N(r)], has now been prepared in quantity, shows the expected thermodynamic unfolding properties and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP. In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K/a = 1.4 x 10(5) M(-1) and delta H = +8.8 kcal x mol(-1). Both values are comparable to those for HPr binding to intact EI. Fluorescence anisotropy [dansyl-EI-N(r)] and gel filtration of EI-N(r) show that it does not dimerize. These results emphasize the role of EI-C in dimerization and the regulation of intact EI.