Enzyme Kinetic Assays Research Articles

Tissue Dissociation Enzyme Neutral Protease Assessment

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate–conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.

Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease

The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41⋯Cys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.

Quimiometria como ferramenta analitica para definição das condiçoes de ensaio da enzima peroxidase de soja

Enzimas Peroxidases são heme-proteínas encontradas nos diferentes organismos vivos, especialmente vegetais, apresentam importante papel fisiológico/bioquímico como proteção contra microorganismos invasores. A soja, um dos mais importantes produtos para o agronegócio brasileiro apresenta na casca de suas sementes (subproduto) alta atividade de peroxidase, denominada soybean peroxidase,com potencial de utilização em métodos analíticos clínicos. A proposta do trabalho foi aplicar o planejamento fatorial para otimização das condições extração da enzima, definição das condições ótimas de atividade (pH e temperatura), utilizando metodologia de superfície de resposta. Os dados obtidos com clara definição foram: i) extração em pó cetonico, ii) meio reacional: pH 3,3, volume da amostra contendo a enzima 330 µL - 340 µL, peróxido de hidrogênio 4,2 mmol.L-1 150 µL, tempo de reação 20 segundos, temperatura 50º C, substrato guaiacol 30mmol.L-1 300 µL, e 0,1 mol.L-1 de NaCl. O uso da dessa metodologia para definição das condições de extração e estudos cinético-enzimáticos da peroxidase de soja foram eficientes e mais precisos, comparado a metodologia de variações/repetições (tentativa e erro).

Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K(d)(app) = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.

Effects of metformin onplasma lactic acid level in patients with type 2 diabetes

：One hundred andthirty-six adult patients with type 2 diabetes ( ≥40 years, body mass index > 24) were assigned to thelow-dose Mefformin, high-dose Mefformin, and control groups. Plasma lactic acid level wasdetected with ultraviolet enzyme kinetics assay. Our data showed that plasma lactic acidof the low-dose or high-dose mefformin group was significantly higher than the controlgroup (2.314 vs. 2.511, P 2. 2 mmol/L) was found in some patients ( Mefformin group vs. Control group: X2 =7.43, P 5.0 mmol/L was found. Insulin resistanceindex and hemoglobin Alc were similar between the 3 groups. Thus, mefformin may notincrease the risk of lactic acidosis in diabetic patients with normal cardiorespiratoryand renal function. Key words: Diabetes mellitus,type 2; Metformin

Crystallographic and activity‐based evidence for thumb flexibility and its relevance in glycoside hydrolase family 11 xylanases

Enzyme intramolecular mobility and conformational changes of loops in particular play a significant role in biocatalysis. In this respect, the highly conserved thumb loop of glycoside hydrolase family (GH) 11 xylanases is an intriguing and characteristic structural element, of which the true dynamic nature and function in catalysis is still unknown. Crystallographic analysis of the structure of a Bacillus subtilis xylanase A mutant, found as a dimer in an asymmetric unit, revealed that the thumb region can adopt an extended conformation, which is stabilized in the crystal lattice through intermolecular contacts. In contrast to the closed thumb conformation of GH11 xylanases and the previously observed small conformational changes upon substrate binding, a relocation of the tip of the thumb of more than 15 A was observed. Site-directed mutagenesis of five thumb residues, including putative hinge point residues, and enzyme kinetics assays showed that Arg112, Asn114, and Thr126 play a role in the open-close thumb movement. Replacement of Arg112 by glycine or proline caused a strong decrease of turnover numbers and elevated Michaelis constants on xylan. Mutant N114P hindered thumb movement, provoking a fourfold decrease of turnover numbers and a sharp rise in Michaelis constants, whereas the proline mutant of Thr126 displayed an increase in specific activity. The observation that extensive thumb opening is possible combined with the kinetic data suggests that the thumb plays a crucial role in both binding of substrate and release of product from the active site.

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α- d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α- d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower p K a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.

Identification of non-catalytic matrix metalloproteinase 1 residues that participate in collagenolysis.

Abstract Abstract #2063 Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity, and at least one of these (MMP-1) has been strongly implicated in the progression of breast cancer. The present study has utilized triple-helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry (HDX MS) in combination with site-specific mutagenesis and enzyme kinetic assays have allowed us to identify non-catalytic sites in MMP-1 that participate in collagenolysis. As we have recently demonstrated [J. Am. Chem. Soc. 129, 10408 (2007)], non-catalytic sites can be utilized to create THP inhibitors that target proteases implicated in cancer progression while sparing proteases with host-beneficial functions. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2063.

Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases

BackgroundPurine nucleoside phosphorylase (PNP) is central to purine salvage mechanisms in Plasmodium parasites, the causative agents of malaria. Most human malaria results from infection either by Plasmodium falciparum (Pf), the deadliest form of the parasite, or by the widespread Plasmodium vivax (Pv). Whereas the PNP enzyme from Pf has previously been studied in detail, despite the prevalence of Pv little is known about many of the key metabolic enzymes from this parasite, including PvPNP.ResultsThe crystal structure of PvPNP is described and is seen to have many features in common with the previously reported structure of PfPNP. In particular, the composition and conformations of the active site regions are virtually identical. The crystal structure of a complex of PfPNP co-crystallised with inosine and arsenate is also described, and is found to contain a mixture of products and reactants – hypoxanthine, ribose and arsenate. The ribose C1' in this hybrid complex lies close to the expected point of symmetry along the PNP reaction coordinate, consistent with a conformation between the transition and product states. These two Plasmodium PNP structures confirm the similarity of structure and mechanism of these enzymes, which are also confirmed in enzyme kinetic assays using an array of substrates. These reveal an unusual form of substrate activation by 2'-deoxyinosine of PvPNP, but not PfPNP.ConclusionThe close similarity of the Pf and Pv PNP structures allows characteristic features to be identified that differentiate the Apicomplexa PNPs from the human host enzyme. This similarity also suggests there should be a high level of cross-reactivity for compounds designed to inhibit either of these molecular targets. However, despite these similarities, there are also small differences in the activities of the two Plasmodium enzymes.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Aliskiren Accumulates in Renin Secretory Granules and Binds Plasma Prorenin

The vascular effects of aliskiren last longer than expected based on its half life, and this renin inhibitor has been reported to cause a greater renin rise than other renin-angiotensin system blockers. To investigate whether aliskiren accumulation in secretory granules contributes to these phenomena, renin-synthesizing mast cells were incubated with aliskiren, washed, and exposed to forskolin in medium without aliskiren (0.1 to 1000 nmol/L). (Pro)renin concentrations were measured by renin- and prorenin-specific immunoradiometric assays, and renin activity was measured by enzyme-kinetic assay. Without aliskiren, the culture medium predominantly contained prorenin, the cells exclusively stored renin, and forskolin doubled renin release. Aliskiren dose-dependently bound to (pro)renin in the medium and cell lysates and did not alter the effect of forskolin. The aliskiren concentrations required to bind prorenin were 1 to 2 orders of magnitude higher than those needed to bind renin. Blockade of cell lysate renin activity ranged from 27+/-15% to 79+/-5%, and these percentages were identical for the renin that was released by forskolin, indicating that they represented the same renin pool, ie, the renin storage granules. Comparison of renin and prorenin measurements in blood samples obtained from human volunteers treated with aliskiren, both before and after prorenin activation, revealed that <or=30% of prorenin was detected in renin-specific assays. In conclusion, aliskiren accumulates in renin granules, thus allowing long-lasting renin-angiotensin system blockade beyond the half-life of this drug. Aliskiren also binds to prorenin. This allows its detection as renin, and might explain, in part, the renin rise during renin inhibition.

Antioxidant enzymes glutathione peroxidase and glutathione reductase correlate with human embryo cell number

OBJECTIVE: Glutathione peroxidase and glutathione reductase are antioxidant enzymes that assist cells in reducing destructive reactive oxygen species. Glutathione peroxidase expression in the oocyte has been associated with successful oocyte maturation. In this study, we evaluate the impact of these and other antioxidant enzymes in follicular fluid (FF) on human embryo cell number as a surrogate marker of oocyte competence during IVF.DESIGN: Prospective study.MATERIALS AND METHODS: FF from a single 18-20 mm follicle was obtained from 60 women (ages 23-44) undergoing IVF. Serum was also obtained at the time of oocyte retrieval. Antioxidant enzyme activities (glutathione peroxidase, glutathione reductase, glutathione s-transferase, superoxide dismutase) were measured by kinetic enzyme assay. Bivariate associations were evaluated among FF antioxidant enzymes and embryo cell number from the corresponding oocyte, controlling for day of embryo transfer.RESULTS: Glutathione peroxidase, glutathione reductase and glutathione s-transferase correlate with each other in FF (rsp> 0.35, P < 0.01), but only glutathione peroxidase and glutathione s-transferase correlate in serum (rsp > 0.28, P < 0.05) (Table 1). In 31 women with day three embryo transfer, glutathione peroxidase and glutathione reductase correlated positively with embryo cell number. Glutathione s-transferase and superoxide dismutase did not correlate with embryo cell number in a statistically significant fashion (P>0.05).Table 1Antioxidant EnzymeEmbryo Cell Number (n=31)Glutathione peroxidase0.40 (95% CI 0.04, 0.66)Glutathione reductase0.43 (95% CI 0.08, 0.68)Glutathione s-transferase0.01 (95% CI -0.35, 0.37)Superoxide dismutase-0.15 (95% CI -0.48, 0.23) Open table in a new tab OBJECTIVE: Glutathione peroxidase and glutathione reductase are antioxidant enzymes that assist cells in reducing destructive reactive oxygen species. Glutathione peroxidase expression in the oocyte has been associated with successful oocyte maturation. In this study, we evaluate the impact of these and other antioxidant enzymes in follicular fluid (FF) on human embryo cell number as a surrogate marker of oocyte competence during IVF. DESIGN: Prospective study. MATERIALS AND METHODS: FF from a single 18-20 mm follicle was obtained from 60 women (ages 23-44) undergoing IVF. Serum was also obtained at the time of oocyte retrieval. Antioxidant enzyme activities (glutathione peroxidase, glutathione reductase, glutathione s-transferase, superoxide dismutase) were measured by kinetic enzyme assay. Bivariate associations were evaluated among FF antioxidant enzymes and embryo cell number from the corresponding oocyte, controlling for day of embryo transfer. RESULTS: Glutathione peroxidase, glutathione reductase and glutathione s-transferase correlate with each other in FF (rsp> 0.35, P < 0.01), but only glutathione peroxidase and glutathione s-transferase correlate in serum (rsp > 0.28, P < 0.05) (Table 1). In 31 women with day three embryo transfer, glutathione peroxidase and glutathione reductase correlated positively with embryo cell number. Glutathione s-transferase and superoxide dismutase did not correlate with embryo cell number in a statistically significant fashion (P>0.05).

Newly developed renin and prorenin assays and the clinical evaluation of renin inhibitors

The last decade has seen the introduction of renin inhibitors and new plasma renin and prorenin assays, which has led to a better understanding of the tissue renin-angiotensin system. To clarify the consequences of these developments for the methodology and interpretation of measurements of renin and prorenin. The principles and application of the newly developed immunosorbent assays (ISAs) are surveyed and the results are compared with those of enzyme-kinetic assays (EKAs). Angiotensin (Ang) II in cardiac, renal and adrenal tissue is known to originate mainly from locally produced Ang I. Experimental evidence and theoretical considerations show that a simple relation between Ang II receptor occupancy, in tissue micromilieu, and the circulating levels of Ang II or renin may not exist. This supports the clinicians' view that the plasma level of renin tells more about the mechanisms regulating its release into the circulation than about the Ang II-dependency of hypertension. ISAs are a welcome addition to clinical studies of renin inhibitors. By comparing the results of ISAs with those of EKAs, the inhibitor-bound forms of renin and prorenin can be distinguished from the unbound forms. ISAs also provide important information on the molecular basis of prorenin activation. We propose a single kinetic model to incorporate the conformational changes of prorenin induced by cryo-activation and acid-activation, and by binding to renin inhibitors. It explains why renin ISAs can overestimate the rise of renin in response to these drugs, and shows how to deal with this artefact.

Enzyme kinetics assay in ionic liquid-based reaction media by means of Raman spectroscopy and multivariate curve resolution

Enzymatic hydrolysis of p-nitrophenylphosphate by alkaline phosphatase in binary mixtures of water and 1-ethyl-3-methylimidazolium tetrafluoroborate (emimBF 4) was monitored with Raman microspectroscopy. Concentrations of emimBF 4 in the studied ionic liquid/water solvent systems ranged from 0 to 75% v/v. Multivariate curve resolution-alternating least squares (MCR-ALS) was successfully applied to the recorded Raman spectra in order to retrieve the concentration profiles and pure Raman spectra of the different species involved in the reaction. Michaelis–Menten constant ( K M) and maximum rate ( V max) of the reaction were calculated from the initial reaction phase for the different solvent systems studied, to investigate the effect of increasing concentration of the ionic liquid on the kinetic behavior. From this study, it was found that the ionic liquid inhibits the reaction under study decreasing both V max and K M.

Laughter modulates prorenin receptor gene expression in patients with type 2 diabetes

Objective The purpose of this study was to assess whether laughter influences the expression of the receptor gene for prorenin that participates in the progression of diabetic nephropathy. Methods Sixteen normal subjects and 23 patients with type 2 diabetes [12 nephropathy (−) and 11 nephropathy (+)] were recruited to examine the effects of laughter on the modulation of prorenin receptor gene expression. After watching a comedy show, laughter-induced changes in the levels of blood prorenin and the expression of prorenin receptor gene were analyzed by an antibody-activating direct enzyme kinetic assay and by reverse transcriptase polymerase chain reaction, respectively. Results In diabetic patients, laughter decreased the level of blood prorenin [93.4–60.4 ng/l in nephropathy (−) patients, 196.6–166.7 ng/l in nephropathy (+) patients] and up-regulated the prorenin receptor gene [1.49-fold in nephropathy (−) patients, 1.46-fold in nephropathy (+) patients]. No significant changes in the expression of this gene were recognized in normal subjects. Conclusion The beneficial effects of laughter on preventing the exacerbation of diabetic nephropathy are strongly suggested in terms of normalizing the expression of the prorenin receptor gene followed by reducing the level of blood prorenin.

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP) — Immunohistochemical Study of ΔTC-PTP Expressions in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemicaily the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔATC-PTP was expressed in E. coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP (76.92%, 10/13) than adenocarcinoma (57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.

Accuracy and Biological Variation of Human Serum Paraoxonase 1 Activity and Polymorphism (Q192R) by Kinetic Enzyme Assay

Paraoxonase 1 (PON1) phenotype is a better predictor of atherosclerosis risk than are PON1 genetic polymorphisms alone. Larger studies are required to determine the role of PON1 and there is a need for standardized PON1 assays between laboratories. We have adapted 5 enzyme kinetic assays for high-throughput automated analysis of PON1 activity. Using different substrates and reaction conditions, we measured PON1 activity and used activity ratios to identify the PON1 Q192R genetic polymorphisms and assessed the accuracy of the genotype assignments in 79 adult study participants by comparing them with genotypes determined by AlwI restriction enzyme digestion of a 176-bp PCR amplification product from genomic DNA. Imprecision was determined using pooled serum and purified enzyme preparations. Biological variability was estimated by analysis of serial samples from 17 individuals. Variability parameters were compared with total cholesterol as a point of reference to a recognized biomarker of coronary heart disease risk. Salt stimulation and inhibition ratios were 97.4% and 94.7% correct in assigning Q192R genotype, respectively. Analytical imprecision (CV) was 1.0%-3.0% for phenylacetate and paraoxon substrate assays and 3.0%-8.0% for the para-nitrophenylacetate substrate assays. Combination of the 2 ratios into a double ratio resulted in 100% correct genotype classification. The described methods for measurement of PON1 activity and accurate genotype assignment are rapid and have potential to facilitate the efficient investigation of PON1 status in clinical and epidemiological studies.

Phospholamban Inhibits Ca-ATPase Conformational Changes Involving the E2 Intermediate

We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 --> E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.

Prorenin Levels in Retinopathy of Prematurity

To evaluate serum prorenin levels in preterm infants with or without retinopathy of prematurity (ROP). Noncomparative retrospective case control study. Serum prorenin levels in children with a gestational age younger than 36 weeks were measured with antibody-activating direct enzyme kinetic assay. The mean prorenin concentrations were higher in preterm infants with ROP than in infants without ROP at every time point: from 26 to 30 weeks, 2326 +/- 334 microg/ml (mean +/- SE [standard error]) (n = 3) and 1164 +/- 234 microg/ml (n = 3), respectively (P < .05); from 31 to 35 weeks, 1760 +/- 496 microg/ml (n = 10) and 957.1 +/- 139 microg/ml (n = 25) (P < .05); from 36 to 40 weeks, 958.2 +/- 194.2 microg/ml (n = 23) and 470.1 +/- 32.6 microg/ml (n = 67) (P < .01); and from 41 to 45 weeks, 575.8 +/- 148.4 (n = 9) and 385.6 +/- 47.9 (n = 13), which is not significantly different. Prorenin levels in preterm infants can predict which infants will develop ROP.

Effect of Hemolysis on Nonesterified Fatty Acid and β-Hydroxybutyrate Concentrations in Bovine Blood

Nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHB) assays are used for evaluating dairy herds for negative energy balance and subclinical ketosis, respectively. Hemolysis is a common artifact in samples submitted to diagnostic laboratories. The effect of hemolysis on NEFA and BHB in bovine serum was determined. Hemolysis was introduced into 26 serum samples by adding serial dilutions of a red cell hemolysate, prepared by repeated freeze-thawing of EDTA-anticoagulated bovine blood. NEFA, BHB, and degree of hemolysis (hemolytic index) were measured by an automated chemistry analyzer. Two endpoint assays that differed by inclusion of a sample blank were used for NEFA measurement. A kinetic enzymatic assay with 2 reagent sources was used for BHB measurement. The assessed methods yielded similar NEFA or BHB results in baseline, nonhemolyzed samples (median NEFA: 0.25 mEq/L, median BHB: 3 mg/dL, median hemolytic index: 8 units). NEFA results were adversely affected by hemolysis, with values increasing significantly with higher degrees of hemolysis. Median values increased above a critical medical decision limit (0.40 mEq/L) at a hemolytic index of 506 units (marked hemolysis). This increase was prevented by inclusion of a sample blank. Result interpretation was affected in individual animals when samples were moderately hemolyzed (median hemolytic index: 258 units). In contrast, BHB results were unaffected by hemolysis with either reagent source. Thus, assays for measuring NEFAs should include a sample blank and NEFA results should not be interpreted in moderately to markedly hemolyzed bovine samples, because result accuracy cannot be assured.