Biomedical subjects

A Barth

Publications and source records attributed to A Barth.

At least 37 records · Page 2Linked to original sources

PubMed2001

Long-term follow-up of an epidemiologically defined cohort of patients with Tourette syndrome.

The goal of this study was to collect prospective longitudinal information on the development of an epidemiologically defined cohort of patients with Tourette syndrome. These data may improve prognostic understanding of the condition. This information will also be important for specification of an adult phenotype for genetic marker studies. A prospective longitudinal cohort study was conducted. Fifty-four of 73 patients from our 1986 prevalence study of Tourette syndrome in North Dakota school-aged children were eligible for inclusion. The subjects were diagnosed in 1984 and 1985. We were able to interview 39 of 54 eligible patients for 507 person-years of follow-up. For the cohort, tic severity declined by 59%, global assessment of functioning improved by 50%, and the average number of comorbidities decreased by 42%. Forty-four percent of patients were essentially symptom free at follow-up. Only 22% were on medication as adults. Tourette syndrome is a developmental neuropsychiatric disorder with a long-term course that is favorable for most patients. Males demonstrated substantially more variability in improvement but overall demonstrated more improvement than females.

Adolescent↗

PubMed2001

Imaging tumors with peptide-based radioligands.

Regulatory peptides are small, readily diffusable and potent natural substances with a wide spectrum of receptor-mediated actions in humans. High affinity receptors for these peptides are (over-) expressed in many neoplasms, and these receptors may represent, therefore, new molecular targets for cancer diagnosis and therapy. This review aims to give an overview of the peptide-based radiopharmaceuticals which are presently already commercially available or which are in advanced stages of their clinical testing so that their broader availability is anticipated soon. Physiologically, these peptides bind to and act through G protein-coupled receptors in the cell membrane. Historically, somatostatin analogs are the first class of receptor binding peptides having gained clinical application. 111In-DTPA-[D-Phe1]-octreotide is the first and only radiopeptide which has obtained regulatory approval in Europe and the United States to date. Extensive clinical studies involving several thousands of patients have shown that the major clinical application of somatostatin receptor scintigraphy is the detection and the staging of gastroenteropancreatic neuroendocrine tumors (carcinoids). In these tumors, octreotide scintigraphy is superior to any other staging method. However, its sensitivity and accuracy in other, more frequent neoplasms is limited. Radiolabeled vasoactive intestinal peptide (VIP) has been shown to visualize the majority of gastrointestinal adenocarcinomas, as well as some neuroendocrine tumors, including insulinomas (the latter being often missed by somatostatin receptor scintigraphy). Due to the outstanding diagnostic accuracy of the pentagastrin test in detecting the presence, persistence, or recurrence of medullary thyroid cancer (MTC), we postulated the expression of the corresponding (ie. cholecystokinin [CCK-] -B) receptor type in human MTC. This receptor is also widely expressed on human small-cell lung cancer. Indeed, 111In-labeled DTPA derivatives of gastrin showed excellent targeting of CCK-B receptor expressing tissues in animals and patients. A variety of further peptide-based radioligands, e.g. among many others, gastrin-releasing peptide/bombesin, neurotensin, substance-P, pan-somatostatin (somatostatin derivatives which bind to all five receptor subtypes) or glucagon-like peptide-1 (glp-1) analogs (the latter for the specific detection of insulinomas), is currently under development. Summarizing, radiolabeled regulatory peptides have opened new horizons in nuclear oncology for diagnosis (and potential internal radionuclide therapy). Future work will probably reveal a multitude of novel potentially clinically useful peptide-based radioligands.

Humans↗

PubMed2000

[Results and role of carotid endarterectomy].

BACKGROUND: The indications for carotid endarterectomy have been defined on the basis of several large prospective randomised trials. Today this operation is widely performed as primary and secondary stroke prevention in patients with high grade carotid artery stenosis. Permanent quality control of the surgical results is mandatory to ensure optimal stroke prevention and further reduce perioperative complications. METHODS: In this retrospective study we analyse the surgical results of 272 consecutive carotid endarterectomies performed in 260 patients with special emphasis on the prevention of intraoperative brain ischaemia. Patients were operated on in general anaesthesia and moderate hypothermia. Before clamping the arteries at the neck, a fast-acting barbiturate or propofol was administered intravenously to obtain burst suppression on the EEG. Transcranial Doppler sonography allowed continuous intraoperative monitoring of brain perfusion and detection of emboli. Intraoperative shunting was performed only when the collateral circulation was insufficient. RESULTS: The postoperative course was uneventful in 249 endarterectomies (91.5%). The internal carotid artery was sonographically recanalised at hospital discharge and at 6-week follow-up in all 249 cases. Ischaemic cerebral complications were observed in 7 patients (2.6%): minor reversible brain ischaemia in 4 (1.5%), and major brain ischaemia with infarction in 3 (1.1%). Three patients died in the perioperative period, representing a mortality rate of 1.1% in this series. The overall combined stroke and death rate was 3.7%. The combined major stroke and death rate was 2.2%. Various non cerebral complications occurred in 13 patients (4.8%). CONCLUSIONS: These results confirm that endarterectomy is a safe and efficient treatment for atherosclerotic carotid stenosis at the neck. Very low complication rates can be attained by non-invasive diagnostic methods combined with intraoperative monitoring of cerebral blood flow velocity. New upcoming endovascular therapies such as percutaneous angioplasty and stenting need to be compared with these current surgical results with regard not only to perioperative morbidity and mortality but also patency rate, restenosis, and intracerebral blood flow restoration.

Adult↗

PubMed2000

Substrate binding and enzyme function investigated by infrared spectroscopy.

Protein conformational changes triggered by molecule binding are increasingly investigated by infrared spectroscopy often using caged compounds. Several examples of molecule-protein recognition studies are given, which focus on nucleotide binding to proteins. The investigation of enzyme mechanisms is illustrated in detail using the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane as an example. It is shown that infrared spectroscopy provides valuable information on general aspects of enzyme function as well as on molecular details of molecule-protein interactions and the mechanism of catalysis.

Calcium-Transporting ATPases↗

PubMed2000

[Pseudostenocardia due to exposure to "electrosmog"].

HISTORY AND REASON FOR ADMISSION: A 44-year-old man had anginal-like complaints. He was convinced that cardiac problems had been caused by electromagnetic fields. INVESTIGATIONS: Apart from hypertension and hyperlipidaemia there were no other significant findings. ECG, ergometry Holter-ECG were normal. EXPERIMENT: We tried to verify with a double-blind provocation test if the patient's complaints had been caused by electromagnetic fields. The patient was exposed to an electric instrument without being aware whether it was switched on or off. He was questioned about his perception of the presence of an electric current. CONCLUSION: The results of the test showed that there was no causal relation between the patient's angina-like-symptoms and electromagnetic fields. The phenomenon of electrosensitivity was probably caused by psychological mechanisms.

Adult↗

PubMed2000

Influence of subchronic administration of oestrone-3-O-sulphamate on oestrone sulphatase activity in liver, spleen and white blood cells of ovariectomized rats.

Inhibition of oestrone sulphatase followed by oestrogen removal from tumour cells may be a new form of endocrine therapy of breast cancer in women. We investigated the inhibitory effect of the subchronic administration of oestrone-3-O-sulphamate (EMATE), a steroid sulphatase inhibitor, to ovariectomized rats, to evaluate this method for testing new nonsteroidal inhibitors. EMATE in DMSO was administered both orally and subcutaneously (s.c.) for 7 days at doses of 0.5 and 2.5 mg/kg. In addition the rats were injected s.c. with 0.5 mg oestrone sulphate/kg 26 and 2 h before decapitation under ether anaesthesia. Oestrone sulphatase activity (ESA) was measured radiometrically using [3H]oestrone sulphate as substrate for desulphuration in white blood cells, liver homogenate, microsomes and spleen homogenate. ESA in liver microsomes was found to be nearly 40 times higher than in white blood cells while in spleen ESA was nearly half of that found in liver homogenates and white blood cells. ESA can be inhibited by EMATE down to 50-1.5% of control activity depending on the dose and administration route. The inhibition was in the order, liver homogenate < spleen < liver microsomes < white blood cells, and was more pronounced after s.c. administration of the inhibitor than after oral administration. Ovariectomy was found to be not necessary for oestrone sulphatase-inhibiting studies. Two sequential s.c. injections of oestrone sulphate enhanced the enzyme activities significantly in liver and white blood cells, but not in spleen. In conclusion, white blood cells and liver microsomes of intact female rats can be used for ESA-inhibiting studies. Sulphate-conjugated oestrone can induce oestrone sulphatase in vivo in liver and white blood cells thereby enhancing oestrogen supply in the peripheral organs.

Administration, Oral↗

PubMed2000

Diffusion-weighted MRI for monitoring neurovascular interventions.

Carotid stenting is increasingly considered as treatment for carotid artery disease. A reliable noninvasive method is desirable for assessing the safety of the procedure. Diffusion-weighted MRI (DWI) is sensitive to early brain ischaemia which becoming widely available and might therefore serve this purpose. We prospectively studied 19 patients referred for investigation of carotid artery disease by echo-planar whole-brain DWI before and within 24 h of stenting. The images obtained at a high b value were examined by two independent blinded reviewers for new high-signal areas consistent with ischaemia. We found that 15 patients had no new changes after stenting. One patient showed enlargement of a posterior watershed lesion after the procedure, which correlated with an increase in neurological deficit. Three other patients had presumed small embolic infarcts on DWI; two were asymptomatic and one had weakness at the hand that corresponded to an embolic infarct with a lesion on DWI in the hand notch. There were no false- positive or -negative results on DWI, when compared to clinical findings. DWI is thus a new method that can demonstrate neurologically silent or asymptomatic infants. It can be used to help to assess the safety and efficacy of neurovascular intervention.

Adult↗

PubMed2000

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na(+),K(+)-ATPase.

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.

Adenosine Triphosphate↗

PubMed2000

Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy.

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.

Adenosine Diphosphate↗

PubMed2000

The infrared absorption of amino acid side chains.

Amino acid side chains play fundamental roles in stabilising protein structures and in catalysing enzymatic reactions. These fields are increasingly investigated by infrared spectroscopy at the molecular level. To help the interpretation of the spectra, a review of the infrared absorption of amino acid side chains in H(2)O and 2H(2)O is given. The spectral region of 2600-900cm(-1) is covered.

Amino Acids↗

PubMed2000

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes.

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Administration, Oral↗

PubMed2000

Fine-structure enhancement--assessment of a simple method to resolve overlapping bands in spectra.

A simple mathematical procedure--fine-structure enhancement--has been assessed on its ability to resolve overlapping bands in spectra. Its advantages and limitations have been explored using synthetic and experimental spectra. Fine-structure enhancement involves smoothing the original spectrum, multiplying the smoothed spectrum with a weighting factor and subtracting this spectrum from the original spectrum. As a result, the fine-structure of the original spectrum is enhanced in the processed spectrum and bands that overlap in the original spectrum appear as distinct bands in the processed spectrum. To be resolved by fine-structure enhancement, Lorentzian lines have to be separated by more than their quarter width at half maximum, Gaussian lines by more than their half width at half maximum. A comparison of fine-structure enhancement and Fourier self-deconvolution shows that Fourier self-deconvolution has in theory a higher potential to resolve overlapping bands. However, this depends crucially on the correct choice of the parameters. In practice, when parameters commonly used are chosen for Fourier self-deconvolution, fine-structure enhancement leads to similar results. This is demonstrated at the example of the infrared absorbance spectrum of the protein papain, where the amide I band components could be resolved similarly with both methods. Thus, fine-structure enhancement seems to be a simple alternative to Fourier self-deconvolution that does not require specialised software.

Models, Chemical↗

PubMed2000

Silent cerebral ischemia detected by diffusion-weighted MRI after carotid endarterectomy.

BACKGROUND AND PURPOSE: Small emboli arising from a friable plaque during carotid endarterectomy (CEA) constitute an important risk of perioperative ischemic complications. To evaluate the incidence and significance of silent cerebral ischemic lesions of embolic origin after CEA, we prospectively examined a series of surgical patients with high-grade carotid stenosis by using diffusion-weighted MRI (DWI). We also tried to correlate postoperative ischemic lesions with the occurrence of sonographic cerebral embolic signals, the presence of plaque ulcerations, and the use of intraoperative shunting. METHODS: Of a consecutive series of 53 patients undergoing elective CEA for high-grade carotid stenosis, 48 patients with unchanged postoperative neurological status were prospectively studied with DWI of the brain the day before and the day after the operation. The magnetic resonance images were analyzed by 2 neuroradiologists blinded to the clinical result of the operation. Any new hyperintense signal was interpreted as a postoperative ischemic lesion. RESULTS: Forty-six (95.8%) of 48 patients had unchanged postoperative brain DWI. In 2 patients (4.2%), a new single asymptomatic hyperintense signal was observed on the side of the operation. Both lesions were small and presumably of embolic origin. They were not related to sonographic embolic signals, plaque ulcerations, or intraoperative shunting. CONCLUSIONS: These results suggest that the incidence of silent ischemic brain lesions of embolic origin after CEA is low and does not correlate with the occurrence of intraoperative sonographic microemboli. They confirm that CEA is a safe procedure that carries a low risk of postoperative cerebral events.

Aged↗

PubMed2000

Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.

Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.

Animals↗

PubMed1999

Phosphoenzyme conversion of the sarcoplasmic reticulum Ca(2+)-ATPase. Molecular interpretation of infrared difference spectra.

Time-resolved Fourier transform infrared difference spectra of the phosphoenzyme conversion and Ca(2+) release reaction (Ca(2)E(1)-P --> E(2)-P) of the sarcoplasmic reticulum Ca(2+)-ATPase were recorded at pH 7 and 1 degrees C in H(2)O and (2)H(2)O. In the amide I spectral region, the spectra indicate backbone conformational changes preserving conformational changes of the preceding phosphorylation step. beta-sheet or turn structures (band at 1685 cm(-1)) and alpha-helical structures (band at 1653 cm(-1)) seem to be involved. Spectra of the model compound EDTA for Ca(2+) chelation indicate the assignment of bands at 1570, 1554, 1411 and 1399 cm(-1) to Ca(2+) chelating Asp and Glu carboxylate groups partially shielded from the aqueous environment. In addition, an E(2)-P band at 1638 cm(-1) has been tentatively assigned to a carboxylate group in a special environment. A Tyr residue seems to be involved in the reaction (band at 1517 cm(-1) in H(2)O and 1515 cm(-1) in (2)H(2)O). A band at 1192 cm(-1) was shown by isotopic replacement in the gamma-phosphate of ATP to originate from the E(2)-P phosphate group. This is a clear indication that the immediate environment of the phosphoenzyme phosphate group changes in the conversion reaction, altering phosphate geometry and/or electron distribution.

Calcium↗

PubMed1999

Influence of oestrogens on formation of reactive oxygen species in liver microsomes of differently aged male Wistar rats.

Metabolic pathways of oestrogens are the formation of catechol oestrogens (CE; 2- and 4-hydroxy-oestrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. We checked the oestrogens oestradiol (E2), oestradiol valerate (E2V) and ethinyloestradiol (EE2) for formation of reactive oxygen species in vitro and ex vivo in male Wistar rats in dependence on age. In liver microsomes of 10-, 30-, 60- and 270-day-old rats the influence of E2, E2V and EE2 (10(-7)-10(-3) M) on NADPH-Fe(++)-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) was investigated. The same parameters, additionally P450 content and monooxygenase activities were measured in liver 9000 x g supernatants after subchronic administration of the oestrogens (1, 10 mg/kg b. wt. orally). The most important results are the strong inhibitory capacities of the oestrogens in vitro on LPO in the order of E2V < E2 < EE2, most pronounced in 10-, 60- and 270-day-old animals. In microsomes of 30-day-old rats with the highest control LPO the antioxidative effect of the oestrogens was lower. Whereas the H2O2 generation was not changed by E2, enhanced by E2V, but diminished by EE2 in all age groups, CL(LUC) and CL(LUM) were inhibited in the order of E2 < E2V < EE2. Also after subchronical treatment of the rats the antioxidative action of the oestrogens was evident, microsomal LPO was inhibited in the order of E2 < E2V < EE2. All oestrogens inhibited ethylmorphine N-demethylation. But enhanced H2O2 generation and increased CL(LUC) also indicate a formation of reactive oxygen species by these oestrogens. Obviously in vitro the antioxidative phenolic structure of the oestrogens dominates, whereas after in vivo administration the dose- and age-dependent biotransformation produces prooxidative in addition to antioxidative structures.

Aging↗

PubMed1998

Specificity and symmetry in the interaction of calmodulin domains with the skeletal muscle myosin light chain kinase target sequence.

The specificity of interaction of the isolated N- and C-terminal domains of calmodulin with peptide WFFp (Ac-KRRWKKNFIAVSAANRFK-amide) and variants of the target sequence of skeletal muscle myosin light chain kinase was investigated using CD and fluorescence. Titrations show that two molecules of either domain bind to 18-residue target peptides. For WFFp, the C-domain binds with 4-fold higher affinity to the native compared with the non-native site; the N-domain shows similar affinity for either site. The selectivity of the C-domain suggests that it promotes occupancy of the correct binding site for intact calmodulin on the target sequence. Far UV CD spectra show the extra helicity induced in forming the 2:1 C-domain-peptide or the 1:1:1 C-domain-N-domain-peptide complex is similar to that induced by calmodulin itself; binding of the C-domain to the Trp-4 site is essential for developing the full helicity. Calmodulin-MLCK-peptide complexes show an approximate two-fold rotational relationship between the two highly homologous domains, and the 2:1 C (or N)-domain-peptide complexes evidently have a similar rotational symmetry. This implies that a given domain can bind sequences with opposite peptide polarities, significantly increasing the possible range of conformations of calmodulin in its complexes, and extending the versatility and diversity of calmodulin-target interactions.

Amino Acid Sequence↗

PubMed1998

Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the 1La and 1Lb component spectra.

Near uv CD spectra of Trp residues in proteins frequently show a complex line shape deriving from the overlap of 1La and 1Lb electronic transitions. This study presents an original empirical method of resolving these components, based on the near uv CD spectra of well-defined complexes of calmodulin domains with target peptides containing a single Trp residue and derived from the skeletal muscle myosin light chain kinase target sequence. Spectra of 4 complexes were used to obtain the 1La and 1Lb component spectra that were then used to analyze further complexes. The broad and featureless 1La spectrum is centered at 279 nm, the 1Lb spectrum shows vibrational fine structure with maxima at 274.9, 281.5, and 289.8 nm. The CD spectrum of most complexes could successfully be fitted with one 1La and one 1Lb spectrum, the 1Lb spectrum being negative for all complexes but the 1La spectrum showing either positive or negative sign. Spectra of some complexes, however, failed to be adequately represented by only one 1La and one 1Lb spectrum. Instead, they could be fitted with one 1Lb spectrum and two 1La spectra with different sign and position. The method is successful in identifying and quantitating the relative intensities of a two-component system, consistent with a single conformation for tryptophan in a protein, and provides a simple indication of cases where a more complicated explanation is required.

Animals↗