Influence of left bundle branch block on left ventricular volumes, ejection fraction and regional wall motion

Background. Left ventricular volumes, ejection fraction and regional wall motion are cardiac parameters which provide valuable information for patient management in a large variety of cardiac conditions. Differences in regional wall motion are of relevance in the field of cardiac resynchronisation therapy. We quantified three-dimensional echocardiographic measurements of left ventricular volumes, ejection and regional wall motion (e.g. expressed as systolic dyssynchrony index (SDI)) in two patient cohorts: patients with normal conduction and patients with complete left bundle branch block. Methods. Thirty-five patients scheduled for routine cardiac examination underwent three-dimensional echocardiography: 23 patients with normal conduction and 12 patients with a complete left bundle branch block. Full-volume datasets were analysed and end-systolic volume (ESV), end-diastolic volume (EDV) and ejection fraction (EF) were obtained. SDI was derived from the standard deviation of the measured times to reach minimal regional volume for each of the 16 segments of the left ventricle. Results. A significant difference was observed in left ventricular volumes, ejection fraction and SDI between the two groups. Patients with complete left bundle branch block showed higher EDV (p=0.025) and ESV (p<0.01) and a lower EF (p<0.01) than patients with normal conduction. SDI is significantly higher in patients with complete left bundle branch block (p=0.004) expressing a higher amount of ventricular dyssynchrony. Intraobserver variability showed excellent correlation coefficients: r=0.99 for EDV, ESV and SDI and r=0.98 for EF. Conclusion. Three-dimensional echocardiography is a feasible and reproducible method for the quantification of left ventricular volumes, left ventricular ejection fraction and regional wall motion. Differences can be assessed between normal patients and patients with left bundle branch block. (Neth Heart J 2007;15:89-94.)     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Electrocardiogram for Predicting Cardiac Functional Recovery

To investigate if the 12-lead resting electrocardiogram (ECG) is a predictor of left ventricular (LV) functional recovery after revascularization of chronic total coronary artery occlusions (CTO). Revascularization was performed in 58 CTO patients who had impaired regional wall motion. The 12-lead resting ECG was used to evaluate Q-wave, QT dispersion, and other parameters. Pre- and postoperative LV regional wall motions were evaluated by real-time three-dimensional echocardiography (RT-3DE). In patients with non-Q-wave, the wall motion score index (WMSI) was dropped from 1.56 ± 0.31 to 1.12 ± 0.21 (P < 0.05), while there was no significant changes (1.73 ± 0.12 and 1.59 ± 0.23, P > 0.05) for WMSI in patients with Q-wave. Preoperative non-Q-wave at baseline was predicted recovery with 88 % sensitivity and 68 % specificity. Positive predictive value for recovery was 67 % in patients with non-Q-wave. The presence of Q-wave can predict non-recovery of the regional wall motion with 68 % sensitivity and 88 % specificity. For CTO patients treated by revascularization, recovery can be predicted reliably through the analysis of pathological Q-wave on the 12-lead resting ECG.     

Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein

During genome sequence analysis of Rhodobacter capsulatus, nearby open reading frames were found that encode a photoactive yellow protein (PYP) and a hypothetical biosynthetic enzyme for its chromophore, a tyrosine ammonia lyase (TAL). We isolated the TAL gene, overproduced the recombinant protein in Escherichia coli, and after purification analyzed the enzyme for its activity. The catalytic efficiency for tyrosine was shown to be approximately 150 times larger than for phenylalanine, suggesting that the enzyme could in fact be involved in biosynthesis of the PYP chromophore. To our knowledge it is the first time this type of enzyme has been found in bacteria.     

Percutaneous balloon mitral valvuloplasty in patients with severe mitral stenosis and low transmitral diastolic pressure gradient

An elevated left atrial pressure and high diastolic pressure gradient (DPG) across the mitral valve are the major hemodynamic abnormalities in mitral stenosis (MS). However, a subgroup of patients with severe MS is characterized by low initial DPG. The authors reviewed the clinical, echocardiographic and hemodynamic data as well as procedural results in 180 patients who underwent percutaneous balloon mitral valvuloplasty (PBMV). An initial mean DPG > 10 mmHg was found in 144 patients (80%) (group A) and mean DPG < or = 10 mmHg in 36 patients (20%) (group B). Patients in group A had higher left ventricular ejection fraction (LVEF) than in group B (61 +/- 5% versus 42 +/- 6%, respectively) and higher cardiac index (2.8 +/- 0.4 versus 2.0 +/- 0.3 l/min/m(2) ). In group B 12 patients (33%) had normal LVEF, whereas 24/36 (67%) had reduced LVEF. All the latter had wall motion abnormalities on ventriculography. Unlike group A, intraprocedural echocardiography was essential for monitoring and evaluating immediate results of PBMV in group B. On follow-up of three years, 75% of group A patients and 55% in group B were in functional class I (p < 0.05). PBMV did not significantly improve symptoms in patients in group B who had preprocedure LVEF < or = 35%.     

Radionuclide angiocardiography,a noninvasive method for evaluating left ventricular ejection fraction and regional wall motion; comparison with contrast left ventricular angiography.

The efficacy of gated synchronous acquisition (GSA) after cardiac blood pool labelling in assessing left ventricular function (ejection fraction and regional wall motion) was evaluated in 31 patients within 24 hours of contrast left ventricular angiography. With the R-wave of the electrocardiogram as a physiologic marker, radionuclide data recorded into an on-line computer allowed construction of cardiac blood pool images during sequential periods of the cardiac cycle. The images, of high count density, have good spatial resolution and can be viewed repetitively in real time in a cine mode. The ejection fractions calculated from the left ventricular time-activity curves corrected for background activity correlated well with the ejection fractions determined from dimension analysis of the contrast left ventricular angiograms (r = 0.87). The results were highly reproducible (r = 0.97). Results of analysis of left ventricular wall motion were similar with the two types of angiograms in 26 of the 31 subjects. GSA is a simple, safe means of evaluating left ventricular ejection fraction and regional wall motion noninvasively.     

Effect of right ventricular hypertrophy on cardiac performance and the relation between right ventricular systolic peak pressure and end-systolic volume

To investigate the role of hypertrophy of the right ventricle upon right heart performance and the significance of the peak systolic pressure/end-systolic volume (P/V) ratio in terms of right ventricular systolic performance, simultaneous measurements of radionuclide ventriculograms and central hemodynamics were done in 32 patients with chronic obstructive pulmonary disease. In 26 of the patients (80%) technically adequate two-dimensional echocardiograms could be performed. In the subset of patients with increased (greater than or equal to 6 mm) right ventricular end-diastolic wall thickness no relationship between pulmonary artery pressure and right ventricular ejection fraction (RVEF) existed in comparison with the remaining patients. P/V indices and cardiac output were not decreased. Considering the patients, whose P/V ratio did not increase from rest to exercise, RVEF decreased highly significantly more than in the remaining patients. The ratio of wall thickness and end-diastolic radius as determinant of peak systolic stress was significantly decreased in these patients compared with the remaining patients. In the patients with right ventricular hypertrophy despite significantly higher values of pulmonary artery pressures and resistances, the afterload in terms of systolic wall stress is markedly reduced. We conclude that in the hypertrophic state, right ventricular performance is not impaired despite decreased RVEF values. In the patients whose P/V ratio does not increase from rest to exercise, an inappropriate high peak systolic wall stress may exist both due to inadequate wall thickness and increased diameter of the right ventricle. The role of P/V in terms of prognosis and development of decompensated right heart failure remains undetermined.     

Predicting the signaling state of photoactive yellow protein

As a bacterial blue light sensor the photoactive yellow protein (PYP) undergoes conformational changes upon signal transduction. The absorption of a photon triggers a series of events that are initially localized around the protein chromophore, extends to encompass the whole protein within microseconds, and leads to the formation of the transient pB signaling state. We study the formation of this signaling state pB by molecular simulation and predict its solution structure. Conventional straightforward molecular dynamics is not able to address this formation process due to the long (microsecond) timescales involved, which are (partially) caused by the presence of free energy barriers between the metastable states. To overcome these barriers, we employed the parallel tempering (or replica exchange) method, thus enabling us to predict qualitatively the formation of the PYP signaling state pB. In contrast to the receptor state pG of PYP, the characteristics of this predicted pB structure include a wide open chromophore-binding pocket, with the chromophore and Glu(46) fully solvent-exposed. In addition, loss of alpha-helical structure occurs, caused by the opening motion of the chromophore-binding pocket and the disruptive interaction of the negatively charged Glu(46) with the backbone atoms in the hydrophobic core of the N-terminal cap. Recent NMR experiments agree very well with these predictions.     

心脏磁共振成像评价缺血性心肌病的临床应用价值

目的:评估心脏磁共振(cardiac magnetic resonance,CMR)功能成像在缺血性心肌病临床诊断中的价值。方法:使用飞利浦3.0T磁共振仪,对32例临床确诊的缺血性心肌病患者进行CMR平扫及钆对比剂动态增强扫描。应用Cardiac MR Analysis软件进行相关后处理分析,计算左室射血分数、室壁增厚率等心功能参数并与超声心动图检查结果相比较。采用17节段分段法分析心脏形态学、心肌组织运动、局部灌注、延迟增强等特点,评价其临床应用价值。结果:所有患者的心功能参数均降低,包括左室射血分数、每搏输出量、心输出量和室壁增厚率,心脏磁共振和超声心动图的测量结果并无明显差异(49%±5.3%vs 52%±8.2%;42.8 mL±8.9 mLvs 45.7 mL±10.6 mL; 3.5 L/min±0.6 L/min vs 3.8 L/min±0.9 L/min; 28%±4%. vs 31%±6%)(P0.05)。所有患者中存在室壁运动异常的为184段(184/544);其中心肌血流灌注信号减低的有136段(136/184),呈现心肌延迟强化的有98段(98/136)。结论:CMR功能成像对于缺血性心肌病的临床诊疗及预后评估可提供与心肌形态及功能相关的重要信息。     

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.     

Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering

It is widely accepted that PYP undergoes global structural changes during the formation of the biologically active intermediate PYP(M). High-angle solution x-ray scattering experiments were performed using PYP variants that lacked the N-terminal 6-, 15-, or 23-amino-acid residues (T6, T15, and T23, respectively) to clarify these structural changes. The scattering profile of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 A(-1) < Q < 0.8 A(-1)). The intensities and positions of the peaks were systematically changed as a result of the N-terminal truncations. These observations and the agreement between the observed scattering profiles and the calculated profiles based on the crystal structure confirm that the high-angle scattering profiles were caused by intramolecular interference and that the structure of the chromophore-binding domain was not affected by the N-terminal truncations. The profiles of the PYP(M) intermediates of the N-terminally truncated PYP variants were significantly different from the profiles of the dark states of these proteins, indicating that substantial conformational rearrangements occur within the chromophore-binding domain during the formation of PYP(M). By use of molecular fluctuation analysis, structural models of the chromophore-binding region of PYP(M) were constructed to reproduce the observed profile of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting beta4 and beta5, and the deformation of the alpha4 helix. High-angle x-ray scattering with molecular fluctuation simulation allows us to derive the structural properties of the transient state of a protein in solution.     

Systolic and diastolic ventricular function in zebrafish embryos: Influence of norepenephrine,MS-222 and temperature

Background  

Zebrafish are increasingly used to study the influences of gene mutation and manipulation on cardiac development, structure and function. In this study, a video edge detection system was used to characterise, continuously, cardiac ventricle function in 2–5 days old zebrafish embryos embedded in 0.6% agar and examined under light microscopy at room temperature (22°C). Using video edge detection software (IonOptix Inc), the motion of a small region of the cardiac ventricle wall was converted to a continuous chart trace allowing analysis of wall motion amplitude (WMA) and myocardial wall velocity during systole (MWVs) and diastole (MWVd).     

Effect of organic anions on the photoreaction of photoactive yellow protein

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.     

Cardiac Complications as Initial Manifestation of Pheochromocytoma: Frequency,Outcome, and Predictors

ObjectiveTo examine the frequency, outcome, and clinical predictors of cardiac complications as the initial manifestation of pheochromocytoma.MethodsThe medical records of all 76 patients with pheochromocytoma or functional paraganglioma treated at Cedars-Sinai Medical Center, Los Angeles, California, from 1995 to 2011 were reviewed. The patients initially presenting with cardiac complications were identified, and their clinical, laboratory, and imaging characteristics were compared with those of the patients presenting with other complaints, especially hypertension and adrenal mass.ResultsOf the 76 patients, 9 (12%) presented with the following: 2 with acute heart failure, 1 with left ventricular thrombus, 3 with myocardial infarction, and 3 with severe arrhythmia. Failure to diagnose pheochromocytoma resulted in unnecessary invasive interventions in 2 patients. Recovery of cardiac function was excellent after resection of the tumor in all patients. In comparison with the 67 patients presenting with other complaints, the 9 with cardiac complications had similar demographics and cardiac risk factors but harbored larger tumors (6.7 ± 0.8 cm versus 4.4 ± 0.3 cm; P = .015) and exhibited higher biochemical marker levels (23.9 ± 9.0-fold versus 11.3 ± 2.4fold; P = .082), longer corrected QT interval (473 ± 8 ms versus 443 ± 6 ms; P = .015), and lower ejection fraction (43% ± 8% versus 66% ± 2%; P = .002).ConclusionIn this study, 12% of patients with pheochromocytoma initially presented with cardiac complications. Patients with large tumors and high levels of biochemical markers were more likely to develop cardiac injury. Our results confirm that the presence of pheochromocytoma should be ruled out in patients with cardiac diseases and features suggesting pheochromocytoma so that unnecessary interventions can be avoided and cardiac recovery can be achieved. (Endocr Pract. 2012;18:483-492)     

Immunoscintigraphy for detecting acute myocardial infarction without electrocardiographic changes.

OBJECTIVE--To establish whether immunoscintigraphy with antibody to myosin may detect acute myocardial infarction without electrocardiographic changes. DESIGN--Prospective study of patients with suspected acute myocardial infarction or unstable angina with cardiac imaging with 111indium myosin antibody, estimation of cardiac enzyme concentrations, electrocardiography, 201thallium imaging, and radionuclide ventriculography. SETTING--Coronary care unit in a district general hospital. PATIENTS--119 Consecutive patients with suspected acute myocardial infarction or unstable angina. Patients with cardiomyopathy, myocarditis, valvular heart disease, myocardial infarction or cardiac surgery in the previous two weeks or with left bundle branch block and women of childbearing age were excluded. RESULTS--Of 75 patients with suspected acute myocardial infarction, seven had no diagnostic electrocardiographic changes despite normal conduction patterns. Immunoscintigraphy with myosin antibody disclosed necrosis in all seven patients, which was localised in regions supplied by diseased coronary arteries in all but one. Six patients had abnormal images on 201thallium imaging, and all seven had abnormal wall motion at the site of antibody uptake. One patient with minimal left main stem and right coronary artery atheroma had uptake of antibody at two discrete sites. CONCLUSIONS--Immunoscintigraphy with antibody to myosin confirms myocardial infarction in the absence of electrocardiographic changes and discloses the site of infarction.     

Cardiac Abnormalities Associated with Pheochromocytoma and other Adrenal Tumors

ObjectiveTo study the specific cardiac abnormalities associated with pheochromocytoma and to suggest a strategy for evaluating cardiac function in patients with pheochromocytoma.MethodsIn this case-control study, we reviewed pathology records of patients seen at Cedars-Sinai Medical Center between 1997 and 2007; patients with adrenal or extra-adrenal pheochromocytoma and those with nonfunctioning benign or malignant adrenal tumors were identified. Patients with functioning adrenal adenomas that secreted cortisol or aldosterone were excluded. Clinical history, imaging, pathology, biochemical test results, electrocardiographic findings, and echocardiographic findings were compared between patients with pheochromocytoma and patients with nonfunctioning adrenal tumors.ResultsThe charts of 22 patients with pheochromocytoma and 35 patients with nonfunctioning adrenal tumors were included. No perioperative mortality was observed. The average age of patients with pheochromocytoma was similar to that of control patients (51.9 ± 3.9 years vs 60.2 ± 2.5 years, respectively), as was the number of patients with known cardiovascular diseases (2 [9%] in the pheochromocytoma group vs 5 [14%] in the control group). Two patients with pheochromocytoma (9%) exhibited myocardial damage. Abnormal electrocardiographic findings were present in 16 patients with pheochromocytoma (73%) and in 17 control patients (49%) (P = .1). QTc was prolonged in patients with pheochromocytoma compared with control patients (448.3 ± 9.7 ms vs 424.7 ± 4.5 ms, respectively; P = .02) and was correlated with levels of norepinephrine and normetanephrine, but not with levels of epinephrine and metanephrine or tumor size. ST-T abnormalities were present in 11 patients with pheochromocytoma (50%) and in 8 control patients (23%) (P = .04). Echocardiographic findings were normal in most patients with pheochromocytoma; abnormal left ventricular wall motion was documented in 3 patients with long QTc.ConclusionsThe specific electrocardiographic findings in patients with pheochromocytoma are prolonged QTc and ST-T abnormalities. Performing an electrocardiogram in patients with pheochromocytoma would be prudent. Echocardiography would be useful to examine LV wall motion in patients with long QTc. Coronary artery disease should be excluded in patients with significant ST- T changes. (Endocr Pract. 2009;15:10-16)     

Evaluation and classification of right ventricular wall motion abnormalities in healthy subjects by 3-tesla cardiovascular magnetic resonance imaging

Background

Right ventricular (RV) shape and function serves as an indicator in several types of heart disease such as arrhythmogenic right ventricular dysplasia (ARVD). However, there is no in-depth knowledge of RV motion, even in healthy subjects. The aim of our study was to provide a quantitative analysis of normal variations in RV wall motion in healthy subjects by cardiac magnetic resonance imaging (CMRI).

Material and methods

The study population consisted of 65 consecutive patients referred for the evaluation of cardiac function by 3 Tesla CMR. Balanced steady-state free-precession images were obtained and areas of disordered RV wall motion were evaluated and classified based on a standardised segmental model for the right ventricle.

Results

In 59 patients (90.8 %) wall motion abnormalities (WMA) of the right ventricle were evident. WMA were predominately detected in the apicolateral segments (72 %) compared with mediolateral (24 %, P < 0.001) and inferolateral segments (4 %, P < 0.001). Dyskinesia was the most frequent wall motion disorder (62.4 %), followed by hypokinesia (20.8 %) and bulging (16.8 %). The mean WMA diameter in the transverse plane (6.4 ± 1.9 mm) was significantly shorter compared with the diameter in the horizontal long-axis (8.1 ± 3.6 mm, P = 0.002) and short-axis plane (10.7 ± 4.6 mm).

Conclusion

WMA of the right ventricle are common. Therefore, one should be aware that these nonpathological wall motion disorders can easily be mistaken for a pathological regional wall motion contraction, particularly in ARVD where to date, clear wall motion criteria are lacking.     

Upregulation of cardiac insulin-like growth factor-I receptor by ACE inhibition after myocardial infarction: potential role in remodeling.

This study evaluated the effects of angiotensin-converting enzyme (ACE) inhibition after myocardial infarction (MI) on cardiac remodeling and gene expression and localization of components (ligands, receptors, and binding proteins) of the cardiac insulin-like growth factor (IGF) system. After ligation of the coronary artery, rats were randomized to vehicle or ACE inhibitor (Captopril, 50 mg/kg/day) for 4 weeks. Blood pressure, cardiac remodeling, and components of the IGF system were localized in the heart using in situ hybridization (ISH) and immunohistochemistry (IHC). The average infarct size was 42%. There were regional differences in the expression of the IGF system after MI, with increased IGF-I mRNA abundance in the border (24-fold) and infarct (12-fold) and increased IGF-binding protein (IGFBP)-3 mRNA in all areas of the failing left ventricle (threefold). Captopril reduced blood pressure, attenuated cardiac remodeling, and caused a threefold increase in IGF-I receptor mRNA and protein in infarct, border zone, and viable myocardium (p<0.01). Captopril had no effect on IGF-I mRNA but further increased IGFBP-3 mRNA and protein in the border zone, (p<0.05). The changes in the cardiac IGF system following MI are highly localized, persist for at least 4 weeks, and can be modulated by ACE inhibition. These data suggest that the benefits of ACE inhibitors in attenuation of cardiac remodeling may be mediated in part through increased expression of the IGF-I receptor sensitizing the myocardium to the positive effects of endogenous IGF-I.     

pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).     

The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins.

A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.