Flow inefficiencies in non-obstructive HCM revealed by kinetic energy and hemodynamic forces on 4D-flow CMR.

Aims: Patients with non-obstructive hypertrophic cardiomyopathy (HCM) exhibit myocardial changes which may cause flow inefficiencies not detectable on echocardiogram. We investigated whether left ventricular (LV) kinetic energy (KE) and hemodynamic forces (HDF) on 4D-flow cardiovascular magnetic resonance (CMR) can provide more sensitive measures of flow in non-obstructive HCM. Methods and results: Ninety participants (70 with non-obstructive HCM and 20 healthy controls) underwent 4D-flow CMR. Patients were categorized as phenotype positive (P+) based on maximum wall thickness (MWT) ≥ 15 mm or ≥13 mm for familial HCM, or pre-hypertrophic sarcomeric variant carriers (P-). LV KE and HDF were computed from 4D-flow CMR. Stroke work was computed using a previously validated non-invasive method. P+ and P- patients and controls had comparable diastolic velocities and LV outflow gradients on echocardiography, LV ejection fraction, and stroke volume on CMR. P+ patients had greater stroke work than P- patients, higher systolic KE compared with controls (5.8 vs. 4.1 mJ, P = 0.0009), and higher late diastolic KE relative to P- patients and controls (2.6 vs. 1.4 vs. 1.9 mJ, P < 0.0001, respectively). MWT was associated with systolic KE (r = 0.5, P < 0.0001) and diastolic KE (r = 0.4, P = 0.005), which also correlated with stroke work. Systolic HDF ratio was increased in P+ patients compared with controls (1.0 vs. 0.8, P = 0.03) and correlated with MWT (r = 0.3, P = 0.004). Diastolic HDF was similar between groups. Sarcomeric variant status was not associated with KE or HDF. Conclusion: Despite normal flow velocities on echocardiography, patients with non-obstructive HCM exhibited greater stroke work, systolic KE and HDF ratio, and late diastolic KE relative to controls. 4D-flow CMR provides more sensitive measures of haemodynamic inefficiencies in HCM, holding promise for clinical trials of novel therapies and clinical surveillance of non-obstructive HCM.

Determinants of kinetic energy of blood flow in the four-chambered heart in athletes and sedentary controls.

The kinetic energy (KE) of intracardiac blood may play an important role in cardiac function. The aims of the present study were to 1) quantify and investigate the determinants of KE, 2) compare the KE expenditure of intracardiac blood between athletes and control subjects, and 3) quantify the amount of KE inside and outside the diastolic vortex. Fourteen athletes and fourteen volunteers underwent cardiac MRI, including four-dimensional phase-contrast sequences. KE was quantified in four chambers, and energy expenditure was calculated by determining the mean KE/cardiac index. Left ventricular (LV) mass was an independent predictor of diastolic LVKE (R(2) = 0.66, P < 0.001), whereas right ventricular (RV) end-diastolic volume was important for diastolic RVKE (R(2) = 0.76, P < 0.001). The mean KE/cardiac index did not differ between groups (control subjects: 0.53 ± 0.14 mJ·l(-1)·min·m(2) and athletes: 0.56 ± 0.21 mJ·l(-1)·min·m(2), P = 0.98). Mean LV diastolic vortex KE made up 70 ± 1% and 73 ± 2% of total LV diastolic KE in athletes and control subjects (P = 0.18). In conclusion, the characteristics of the LV as a pressure pump and the RV as a volume pump are demonstrated as an association between LVKE and LV mass and between RVKE and end-diastolic volume. This also suggests different filling mechanisms where the LV is dependent on diastolic suction, whereas the RV fills with a basal movement of the atrioventricular plane over "stationary" blood. Both groups had similar energy expenditure for intracardiac blood flow, indicating similar pumping efficiency, likely explained by the lower heart rate that cancels the higher KE per heart beat in athletes. The majority of LVKE is found within the LV diastolic vortex, in contrast to earlier findings.

Potential of the HAQ score as clinical indicator suggesting comprehensive multidisciplinary assessments: the Swedish TIRA cohort 8 years after diagnosis of RA.

This study explores the potential of the health assessment questionnaire (HAQ) score as a clinical indicator that can be used to suggest comprehensive multidisciplinary assessments, by relating it to more general aspects of disability. In a cohort of 132 patients with early RA (mean age 55, 68% women), 28 joint count Disease Activity Scores (DAS-28), HAQ, and Short Form 36 (SF-36) scores were registered at annual follow-up visits 8 years after diagnosis. The patients were tentatively sub-grouped into a high-HAQ group (HAQ ≥ 1 at the 8-year follow-up) and a low-HAQ group. The high-HAQ group, comprising 36% of the cohort, had a higher mean HAQ score at inclusion and beyond at all visits compared to the low-HAQ group, and 24% of all individual patients in the high-HAQ group had a HAQ score ≥ 1 at inclusion. Although the DAS-28 improved in both groups, patients in the high-HAQ group also had significantly more persistent disability according to the SF-36: five scales at each follow-up visit and all eight scales at the majority of the visits. Individual RA patients with HAQ ≥ 1 probably have considerable persistent disabilities according to the SF-36. The HAQ score could be used as a clinical indicator suggesting comprehensive multidisciplinary assessments of the components of disability and corresponding interventions, in addition to the established use of HAQ at group levels and in parallel with the medication strategy based on DAS-28.

Differential effects of fluticasone and montelukast on allergen-induced asthma.

Early asthmatic responses (EAR) and late asthmatic responses (LAR) to allergen are induced by the local release of a series of bronchoconstrictor mediators, including leukotrienes and histamine. Both anti-leukotrienes and other anti-asthma drugs, such as inhaled glucocorticoids, have been shown to reduce both EAR and LAR. The aim of the present study was to directly compare the effects of regular treatment with an oral anti-leukotriene, montelukast (Mont; 10 mg once daily, for 8 days), and an inhaled glucocorticoid [fluticasone propionate (FP) 250 microg twice daily for 8 days] on the EAR and LAR to an inhaled allergen challenge. Patients with a documented EAR and LAR at a screening visit were randomized to these treatments, or placebo, in a double-blind, double-dummy, crossover fashion. Allergen challenge at a dose causing both an EAR and LAR was given on the eighth day of treatment. The maximum fall in FEV1 during the EAR was 17.8% during placebo treatment, 8.3% during Mont and 16.3% during FP (P <0.05 for Mont vs placebo). The maximum fall during the EAR was 13.8% during placebo treatment, 11.8% during Mont and 2% during FP treatment (P <0.05 for FP vs placebo and FP vs Mont). PC20 methacholine was significantly higher 24 h after allergen challenge during FP-treatment compared with Mont (P <0.05). Both montelukast and fluticasone reduced the relative amount of sputum eosinophils after allergen compared with placebo treatment. This study shows that anti-leukotrienes are effective to attenuate the EAR, whereas inhaled glucocorticoids are more effective than anti-leukotrienes in attenuating the EARs and improves bronchial hyperresponsiveness to a greater extent. In conclusion, inhaled glucocorticoids have overall greater efficacy than oral anti-leukotrienes to attenuate allergen-induced airway responses in mild asthmatic patients.

Bronchodilation by an inhaled VPAC(2) receptor agonist in patients with stable asthma.

BACKGROUND: The synthetic vasoactive intestinal peptide (VIP) analogue Ro 25-1553 is a selective VIP-PACAP type 2 (VPAC(2)) receptor agonist that causes a bronchodilatory effect in guinea pigs in vivo. The effect of Ro 25-1553 given by inhalation to patients with asthma was studied and compared with that of a long acting beta(2) adrenoceptor agonist. METHODS: Twenty four patients with moderate stable asthma participated in a double blind, randomised, placebo controlled, crossover study. The primary variable was bronchodilatory effect (increase in forced expiratory volume in 1 second, FEV(1)) after inhalation of Ro 25-1553 (100 microg or 600 microg) and formoterol (4.5 microg), respectively. Putative side effects were characterised by monitoring sitting blood pressure, serum potassium, electrocardiography and echocardiography. RESULTS: Inhalation of 600 microg Ro 25-1553 caused a rapid bronchodilatory effect (geometric mean increase in FEV(1) compared with placebo) within 3 minutes of 6% (95% CI 4 to 9), as did inhalation of formoterol (8% (95% CI 5 to 10)). The corresponding maximum bronchodilatory effect during 24 hours was similar for 600 microg Ro 25-1553 (7% (95% CI 4 to 10)) and the reference bronchodilator formoterol (10% (95% CI 7 to 12)). However, for both doses of Ro 25-1553 the bronchodilatory effect was attenuated 5 hours after inhalation whereas formoterol still had a bronchodilatory effect 12 hours after inhalation. Neither Ro 25-1553 nor formoterol produced any clinically relevant side effects. No drug related difference in adverse events was observed. CONCLUSION: Inhalation of a synthetic selective VPAC(2) receptor agonist constitutes a promising approach for bronchodilation in patients with asthma.

Computational, ReactIR-, and NMR-spectroscopic investigations on the chiral formyl anion equivalent N-(alpha-lithiomethylthiomethyl)-4-isopropyl-5,5-diphenyloxazolidin-2-one and related compounds.

The 4-isopropyl-3-methylthiomethyl-5,5-diphenyloxazolidin-2-one is readily lithiated in THF on the exocyclic CH2 group (1 --> 2) to give a synthetically useful chiral nucleophilic formylating reagent. We have now studied the lithiation reaction by ReactIR spectroscopy and the structure of the organolithium reagent by computational methods and by NMR-spectroscopic measurements. The lithiation is complete at -78 degrees C within 90 seconds, and it is accompanied by a decrease of the C=O wavenumber by 50 cm(-1). The NMR data (collected in [D8]THF) give no evidence for 13C,6Li coupling or for aggregation; from DPFGSE-ROE spectra the single diastereoisomer of the lithium compound 2 seen in the NMR spectra (NMR-spectroscopic measurements from -105 to -20 degrees C) is assigned like configuration; the 13C=O signal of the oxazolidinone undergoes a 7 ppm downfield shift upon lithiation (1 --> 2); line-shape analyses of the signals from the diastereotopic CH2 and CMe2 protons in lithiated 4,4-dimethyl-3-methylthiomethyloxazolidin-2-one (model compound 7) reveal a deltaH(double dagger) of 8.9 +/- 0.2 kcal mol(-1) for enantiomerization. The theoretical calculations provide an energy-minimum structure for the lithium compound 2 with coordination of the carbonyl oxygen to lithium, with an antiperiplanar arrangement of the C,Li and S,CH3 bonds, and with relative like configuration of the two stereocenters--in perfect agreement with the conclusions from the IR- and NMR-spectroscopic measurements!

The therapeutic ratio of R-albuterol is comparable with that of RS-albuterol in asthmatic patients.

BACKGROUND: It has been suggested that R-albuterol produces bronchodilation that is comparable with that of racemic albuterol (RS-albuterol) on a 4:1 dose-for-dose basis but systemic side effects on a 2:1 basis, implying better therapeutic ratio for R-albuterol. OBJECTIVE: We sought to carefully compare the bronchodilating and systemic effects of R- and RS-albuterol by using a crossover study design. METHODS: Twenty asthmatic patients (15.1%-28.7% FEV(1) reversibility) were given R-albuterol (6.25-1600 microg), S-albuterol (6.25-1600 microg), RS-albuterol (12.5-3200 microg), or placebo in a crossover, double-blind, placebo-controlled fashion. Cumulative doses were given with a Mefar dosimeter, and FEV(1), heart rate, and plasma K(+) levels were measured 20 minutes after each dose. RESULTS: Both R- and RS-albuterol produced dose-related improvement in FEV(1) and, at higher doses, increased heart rate and decreased plasma K(+) levels. Neither placebo nor S-albuterol had any significant effect. Individual estimates of the potency ratio for R-albuterol/RS-albuterol were calculated and summarized across all subjects. The geometric mean potency ratio for effects on FEV(1) was 1.9 (95% CI, 1.3-2.8), on HR of 1.9 (95% CI, 1.3-2.9), and on K(+) level of 1.7 (95% CI, 1.3-2.1). CONCLUSION: All pharmacologic effects of RS-albuterol reside with the R-enantiomer, and S-albuterol is clinically inactive. The R-albuterol/RS-albuterol potency ratios for local (FEV(1)) and systemic effects (heart rate and K(+)) are similar, suggesting a comparable therapeutic ratio for R-albuterol and RS-albuterol in asthmatic subjects.

Exudation of plasma and production of thromboxane in human bronchi after local bradykinin challenge.

Plasma exudation has been suggested to be an important component of the inflammatory response in asthma. Bradykinin elicits many of the features of asthma, including bronchoconstriction, cough, plasma exudation and mucus secretion. In an attempt to quantify local plasma exudation, we have employed a novel low-trauma technique with the aim of challenging and lavaging a central part of the bronchial tree, by selecting a medium sized bronchus. A fibreoptic bronchoscopy was performed in non-smoking healthy volunteers. The instrument was placed proximally in the right upper lobe bronchus. A plastic catheter, equipped with an inflatable latex balloon, was inflated with air (2-4 cmH2O). A solution (100 microl of either two different concentrations of bradykinin: 0.09 and 0.9 mg ml(-1) or normal saline) was instilled through the catheter and distal to the balloon. Eight minutes later a lavage procedure with 10 ml of saline was performed through the catheter. The procedure was then repeated twice, with the other solutions, but from the lingular and middle lobe bronchi. All solutions were given in a blinded fashion, and two different studies were performed. Lavage concentrations of albumin and IgG were quantified as measurements of plasma exudation. In our first study we found that bradykinin challenge significantly increased concentrations of albumin and IgG. In study two, there was no numeric increase in plasma proteins after local bradykinin challenge, but the concentration of thromboxane was significantly increased in lavages from bradykinin-challenged bronchi. Thus, local bronchial administration of bradykinin has the capacity to induce exudation of large plasma macromolecules into the bronchial lumen, as well as local thromboxane production.

Polymer versus monomer as displacer in immobilized metal affinity chromatography.

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.

Onset of bronchodilation of budesonide/formoterol vs. salmeterol/fluticasone in single inhalers.

Combinations of inhaled glucocorticoids and long-acting beta2-agonists in the same inhaler device have become available in recent years. In this double-blind, randomized, placebo-controlled and crossover study we have evaluated the onset of action of budesonide and formoterol in a single inhaler (Symbicort Turbuhaler) and that of the fixed combination of salmeterol and fluticasone (Seretide Diskus). Thirty patients with a mean FEV1 of 2.54 l (range: 1.48-4.28) and a mean inclusion reversibility in FEV1 of 19.1% were included. Single doses of budesonide/formoterol 160/4.5 microg and 2x (160/4.5) microg, salmeterol/fluticasone 50/250 microg, or placebo were given. Serial measurements of FEV1 were performed over 3 h. The combination of one or two inhalations of budesonide/formoterol showed a faster onset of action than salmeterol/fluticasone, both evaluated as mean FEV1 at 3 min (2.74, 2.75 and 2.56 l respectively P<0.001 for both doses of budesonide/formoterol), or as average FEV1 from 0 to 15 min (2.80, 2.83 and 2.67 l respectively P<0.001 for both doses of budesonide/formoterol). For placebo, mean FEV1 at 3 min was 2.46 l, and the average FEV1 at 0-15 min was 2.50 l. Furthermore, budesonide/formoterol at both doses resulted in higher FEV1 than salmeterol/fluticasone at 3 h. We conclude that the combination of budesonide/formoterol has a faster onset of action than salmeterol/fluticasone.

The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases.

A series of 36 linear and cyclic beta- and gamma-peptides consisting of as few as two, and as many as 15 residues, was offered as substrates to 15 commercially available proteases of bacterial, fungal, and eukaryotic origin, including a beta-lactamase and amidases, as well as most vigorous, nonspecific proteases, such as the 20S proteasome from human erythrocytes. For comparison, an alpha-eicosapeptide and standard substrates of the proteolytic enzymes were included in the investigation. Under conditions of complete cleavage of the alpha-peptide within 15 min the beta- and gamma-peptides were stable for at least 48 h. Inhibition studies with seven beta- and gamma-peptides and alpha-chymotrypsin show that the residual enzyme activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is unchanged within experimental error after incubation for 15 min with the peptide analogues. Thus, beta- and gamma-peptides with proteinogenic side chains, that is, consisting of the singly or doubly homologated natural alpha-amino acids (one or two CH(2) groups inserted in the backbone of each residue), are completely stable to common proteases, without inhibiting their normal activity (as demonstrated for alpha-chymotrypsin). This proteolytic stability of peptides built of homologated amino acids is a prerequisite for their potential use as drugs.

Equivalent therapeutic ratio of salbutamol given by Turbuhaler and Diskus.

Some inhalers have been claimed to give better deposition, resulting in higher efficacy. In a previous study we did not find any evidence of different potency of salbutamol given either via pMDI or Turbuhaler. The aim of the present study was to compare the efficacy and safety of salbutamol given via Diskus or Turbuhaler. Twenty-five asthmatics with step-wise reversible airflow obstruction (total reversibility of at least 15%) were included in a randomized, double-dummy, placebo-controlled cross-over study. On each study day, the patients were given placebo repeatedly, or cumulative doses of 200, 400, 800, 1600 and 3200 microg salbutamol given via either device (double-blind, placebo-controlled). Salbutamol caused a dose-related increase in FEV1 when given by Diskus or Turbuhaler. The improvement in FEV1 was similar regardless of whether salbutamol was given via Diskus or Turbuhaler, at equivalent microgram doses. After a total cumulative dose of 3200 microg, mean FEV1 for Diskus was 2.46 l (change from baseline of 20.5%), for Turbuhaler 2.50 l (change from baseline 24.6%) and for placebo 2.11 l (3% change from baseline). After correcting for different baseline differences, the percentage difference between Diskus and Turbuhaler was -1.8% (P = 0.2). Systemic effects (potassium and heart rate) did not differ between Diskus or Turbuhaler. We conclude that the efficacy of salbutamol given at equivalent microgram doses, as well as side-effects, are comparable when the drug is given via Diskus or Turbuhaler. The present data shows that salbutamol given by these devices have similar therapeutic ratios.

Similar bronchodilation with formoterol delivered by aerolizer or turbuhaler.

BACKGROUND: In many countries, two dry powder formulations of inhaled formoterol are available for clinical use; one uses a single-dose device (Foradil, Aerolizer), and the other uses a multiple-dose device (Oxis, Turbuhaler). OBJECTIVES: To study the bronchodilating effect of formoterol 12 mg when delivered via the Aerolizer and Turbuhaler devices over 12 h. STUDY DESIGN: Randomized, double-blind, placebo controlled crossover study. Forced expiratory volume in one second (FEV1) was monitored during a 12 h period. PATIENTS: Nineteen nonsmoking asthma patients were included in the trial on the basis of reversibility of symptoms in response to inhaled salbutamol (either 200 or 400 mg given cumulatively; minimum reversibility 15%). RESULTS: There were no significant differences between the two dry powder devices regarding the change from baseline of FEV1 over 12 h, the area under the curve of FEV1 over 12 h or the maximum value of FEV1. The improvement in FEV1 with formoterol 12 mg versus placebo was highly significant for both devices. CONCLUSIONS: Formoterol is similarly effective when used as a dry powder when given by either Aerolizer or the Turbuhaler.

Equivalent bronchodilation with salbutamol given via pMDI or turbuhaler.

Several breath-activated multidose powder devices for inhaled anti-asthma drugs are now available. Some of these inhalers have been argued to give higher drug deposition in the airways than conventional pressurized metered dose inhalers (pMDI). The aim of the present study was to compare the efficacy and safety of salbutamol given via pMDI or Turbuhaler (both 100 microgram per inhalation). Adult asthmatic patients of either sex (n = 22) and with reversible airflow obstruction were included in a randomized, placebo-controlled study. On the study days, salbutamol was given with increasing doses (200 to 3,200 microgram cumulative) or placebo, via pMDI or Turbuhaler. A dose-related increase in FEV1 was observed after administration of salbutamol given via either device, versus placebo. The improvement in FEV1 was similar whether salbutamol was given via pMDI or Turbuhaler, at microgram equivalent doses. After a total cumulative dose of 3,200 microgram, mean FEV1 for Turbuhaler was 2.98 (change from baseline of 23.1%), for pMDI 2.93 (change from baseline of 23.6%), and for placebo 2.36 (change from baseline of 0. 42%). Changes in potassium, glucose, and heart rate did not show any significant differences between pMDI and Turbuhaler. We conclude that the efficacy of salbutamol is comparable when the drug is given via the Turbuhaler or pMDI.

Analysis of nonpolar heterocyclic amines in cooked foods and meat extracts using gas chromatography-mass spectrometry.

Heat processing of muscle foods gives rise to the formation of mutagenic and carcinogenic heterocyclic amines, often at ng/g levels. A gas chromatographic-mass spectrometric (GC-MS) technique was introduced for the analysis of nonpolar heterocyclic amines in common cooked meats, pan residues, and meat extracts after solid-phase extraction. The mutagenic heterocyclic amines 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) were identified in several samples in amounts up to 8 ng/g. Also the comutagenic substances 1-methyl-9H-pyrido [3,4-b]indole (harman) and 9H-pyrido[3,4-b]indole (norharman) were detected in the samples in amounts up to almost 200 ng/g. The GC-MS method can be applied without derivatisation of the sample. The technique offers high chromatographic efficiency, yielding detection limits for pure references in the range 0.1-2 ng per injection.

Protein displacement in dye-ligand chromatography using neutral and charged polymers.

Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using differences spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 degrees C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer.

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein.

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54-60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 µmol g(-1) s(-1) protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20-100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with ß-mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase.

Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration.

The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.

Purification of proteins by the use of hydrophobic zeolite Y.

Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH4)2SO4 or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be absorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH4)2SO4 fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5.