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The article presents the postmortem case studies to determine the distribution of methylone. It mentions about a 19-year old male who was died due to cardiac arrest. It also mentions about a 21-year old female who died due to drowning while chasing by the police and a 35-year old female who was found dead on her bed with a cutting wound on neck and gunshot wound at the back of her head. The studies suggest that methylone and Methylenedioxypyrovalerone are more toxic when taken together.

The article presents a study which measures levetiracetam, topiramate, and hydroxycarbazepine in postmortem cases. The study uses acid or neutral drug screening process to quantify the drugs. Results show that the liver and kidney concentrations of the three drugs are on similar order of magnitude as the blood concentrations, however only the levetiracetam concentration in a single case showed a kidney or liver concentration greater than five times the blood concentration.

The article presents a study which investigates the overdose of extended-release oxymorphone hydrochloride tablets which resulted to death. It mentions the use of liquid chromatography, gas chromatography and mass spectrometry on the analysis of the concentrations of opiate in the blood and urine. Results show that the compounds temazepam, doxylamine and dextromethorphan are present in the urine samples.

The article presents a case study that evaluates the two cases of fatal overdoses of calcium channel blocker amlodipine. Postmortem samples were obtained and amlodipine was measured through liquid chromatography-atmospheric pressure photoionization-mass spectrometry. Results revealed that the heart blood concentrations for the two cases were 2.4 and 0.95 milligram per liter and such drug has been quantified in all other tissues.

Abstract: Detection of a drug and its glucuronide metabolite(s) is of great importance in interpretive forensic and clinical toxicology. Until recently, glucuronides were determined by cleavage of the glucuronide with an enzyme (e.g., β-glucuronidase) to yield the parent compound, which was subsequently detected, or via derivatization to a more volatile or detectable analogue. Direct detection of the glucuronide conjugates overcomes the critical limitations of approaches that involve enzymatic cleavage procedures and/or derivatization. This review will focus on direct methods to determine glucuronides of various potentially abused drugs in human biological matrices. More specifically, this review will be restricted to methods that involve liquid chromatography (LC) coupled with various detectors. Papers are presented in chronological order for each compound class to emphasize the evolution of methodology and continued importance of the application. [Copyright &y& Elsevier]

The article presents a case of a 25-year-old white male who was found dead the morning after consuming herbal extracts containing β-carbolines and hallucinogenic tryptamines. No anatomic cause of death was found at autopsy. The medical examiner ruled that the cause of death was hallucinogenic amine intoxication, and the manner of death was undetermined.

Investigates the involvement of the cytoplasmic region of phospholamban in interaction with the calcium-activated ATPase of the sarcoplasmic reticulum (SERCA). Reduction of the affinity of the peptide for the pump by phosphorylation of Ser16; C-terminal regions of both peptides; Segments of the cytoplasmic domain of SERCA.

The use of retinyl palmitate (RP) as an oral positive control agent was demonstrated in CD rats and New Zealand White rabbits in 3 developmental toxicity studies in each species. Based on dose range-finding studies, 1000 or 300 mg/ kg day -1 (i.e., 250,000 and 75,000 IU/kg day -1) were administered by oral gavage to rats and rabbits, respectively, on gestation days (GD) 9 and 10. The vehicle control groups received either 1% methylcellulose/0.2% Tween 80 or deionized distilled water from GD 6 to 15 in rats and from GD 6 to 18 in rabbits. Maternal clinical signs, body weights, and food consumption were evaluated. Necropsy and cesarean-section observations were conducted on GD 20 and 29 in rats and rabbits, respectively. All viable fetuses were weighed, sexed, and externally examined. In rats but not rabbits, maternal toxicity was observed as significantly decreased body weights and food consumption, while fetal toxicity occurred as significantly decreased fetal body weights. RP was teratogenic in both rats and rabbits, producing a spectrum of significantly increased external, skeletal, and visceral fetal anomalies in all RP-treated groups. The observed malformations were typical of those produced by vitamin A, i.e., cleft palate, pinna anomalies, and microphthalmia/anophthalmia. These studies demonstrate that RP is a reproducible and cost-effective positive control agent for use in rat and rabbit developmental toxicity studies. [ABSTRACT FROM AUTHOR]

Reports on the use of pattern recognition methods to classify gas chromatograms of weathered and unweathered jet fuels. Characterization of jet fuels; Use of discriminant functions.

Examines the relationship of plasma buprenorphine and norbuprenorphine concentrations with withdrawal symptomatology during buprenorphine dose induction, maintenance treatment and withdrawal. Identification of the two opioid agonist substitution medications that were approved by the United States Food and Drug Administration; Information on the use of methadone in treating opioid dependence; Methodology used in the examination.

Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy have been used to quantify metal binding to the phosphoprotein extractable from demineralized rat incisor dentine. Paramagnetic cation probes enable identification of the metal binding sites. Cations are able to diffuse across the protein surface while forming a relatively long-lived metal-phosphoprotein complex. The ability of the protein to sequester surface-mobile Ca(II) is discussed in terms of its ability to act as a possible nucleation site for the initial localization of Ca(II) within the dentine matrix. [ABSTRACT FROM AUTHOR]

Evaluates ribavirin's testicular toxicity and reversibility in mice. Dose levels studied; Drug formation; Description of the experimental method used.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard catdesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1 – 44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389 – 397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20 – 41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483 – 578) derived from the N-terminal portion of a 35-kDa cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin- filament regulation in smooth and striated muscle. [ABSTRACT FROM AUTHOR]

The interaction between troponin-I and actin that underlies thin-filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin-I (residues 96-116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375-387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin-I which has inhibitory activity interacts with the N-terminal region of actin, The surface of contact of the inhibitory segment of troponin-I with actin involves two regions of the N-terminal of actin. These are located between residues 1-7 and 19-44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation. [ABSTRACT FROM AUTHOR]

Reports on an analysis of [sup 1]hydrogen-nuclear magnetic resonance spectroscopic techniques of the conformation of the nitrogen terminal segment of the LC1 alkali light chain of rabbit skeletal muscle which has shown that this portion of the molecule adopts a well-defined elongated configuration. Finding that the rod-like feature is a consequence of the Ala/Pro-rich composition and discussion of the functional aspects of such conformational preference in this and similar segments in other proteins.

1HNMR is used to study the solution structure o f vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein binds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially, Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein. [ABSTRACT FROM AUTHOR]

A previous study suggested that small amounts of morphine are metabolically converted to hydromorphone. In the present study, morphine positive urine specimens obtained from a postmortem laboratory and a random urinalysis program were tested for morphine, codeine, hydromorphone, hydrocodone, oxymorphone, and oxycodone to assess the possibility that small amounts of hydromorphone are produced from the metabolism of morphine. The opioids were analyzed by gas chromatography–mass spectrometry as their respective trimethylsilyl derivatives following solid phase extraction. The limit of detection for hydromorphone was 5 ng/mL. A total of 73 morphine positive urine specimens were analyzed, with morphine concentrations ranging from 131 to 297,000 ng/mL. Hydromorphone was present at a concentration ≥5 ng/mL in 36 of these specimens at concentrations ranging from 0.02% to 12% of the morphine concentration. Hydrocodone was not detected in these specimens at the assay detection limit of 25 ng/mL. These results support earlier work suggesting that the detection of hydromorphone in urine specimens does not necessarily mean that exogenous hydromorphone or hydrocodone was used. [ABSTRACT FROM AUTHOR]

The article provides information on the case of a 47-year-old man whose death was caused by an abuse on sevoflurane. The presence of sevoflurane was confirmed using a headspace gas chromatography-mass spectrometry. The concentration of the heart blood sevoflurane was 16 mg/L, and the concentration of the peripheral blood sevoflurane was 8.0 mg/L. When the heart blood was examined, no other drugs or other volatile substances were confirmed. Moreover, cardiac arrhythmia due to sevoflurane toxicity was the cause of his death as ruled out by the examiner.

Abstract: A case is presented of a 54-year-old white female found dead in a secured apartment. Postmortem toxicologic analysis of the heart blood identified acetaminophen (97mg/L), citalopram (0.4mg/L), gabapentin (24mg/L) and metaxalone (21mg/L). The metaxalone concentration is within the range of previously reported fatalities involving metaxalone. The medical examiner ruled that the cause of death was metaxalone and gabapentin intoxication and the manner of death was suicide. [Copyright &y& Elsevier]

Describes a case of oxycodone intoxication in a 10-month old infant. Identification of oxycodone in postmortem specimens using gas chromatography-nitrogen phosphorus detection and confirmation by full scan electron ionization gas chromatography-mass spectrometry; Suggestion that there are serious concerns about the likelihood of drug exposure through consumption of breast milk.

Investigates hydroxycarbazepine distribution in three postmortem cases by researchers from Baltimore, Maryland. Detection of the active metabolite of the anticonvulsant oxacarbazepine in the biological specimens submitted for toxicological analysis; Use of gas chromatography-nitrogen-phosphorus for the detection of the compound; Assertion from the medical examiner that the hydroxycarbazepine was an incidental finding to the cause of death.

Presents an analysis of metformin in antemortem serum and postmortem specimens by a novel high-performance liquid chromatography (HPLC) method and application to an intoxication case. Performance characteristics of the metformin assay; Drugs tested and found not to interfere with the metformin assay; Elimination half-life of metformin.

Comments on the interpretation of analytical findings on urine samples generated by the laboratory by the forensic toxicologist. Role of the identified substances in the cause of death; Assessment of toxicity or impairment in living individuals; Correlation of impairment with drug concentrations.

A letter to the editor is presented in response to comments made to the author's article "A Fatal Intoxication Following the Ingestion of 5-Methoxy-N,N-Dimethyltryptamine in an Ayuhuasca Preparation."

The article presents the case study of a 34-year-old woman with mild exertional dyspnea and interstitial pulmonary lesions. She was admitted to the hospital and several tests were conducted. On physical examination the patient was an obese woman who was comfortable at rest. No rash or lymphadenopathy was found. The head and neck were normal. The lungs were clear, and the heart, breasts, and abdomen were normal. Neurologic examination was negative.

A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT1B/1D receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography–tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident. [ABSTRACT FROM AUTHOR]

Caffeine, 1,3,7-trimethylxanthine, is the most widely consumed legal stimulant. The most potent form is over the counter oral tablets. The estimated average daily adult caffeine consumption is 300 mg. Rarely, serious toxicities such as seizure and cardiac arrhythmias have caused death. Caffeine concentrations of 80 mg/L are considered lethal. Cases investigated over 10 years by the Office of the Chief Medical Examiner for the State of Maryland ( OCME) whose cause of death was solely or in combination due to caffeine intoxication were reviewed. A total of eight adult cases were identified consisting of two suicides and six undetermined manners. The average postmortem caffeine concentration was 140.4 mg/L. This is the largest case series reported to date of lethal caffeine intoxication. Large doses of prescription medications and/or over the counter medications were abused. No overdoses were from consumption of caffeine-containing foods. Comprehensive postmortem toxicological testing should test for caffeine to capture these rare deaths. [ABSTRACT FROM AUTHOR]

Caffeine, 1,3,7-trimethylxanthine, is the most widely consumed legal stimulant. The most potent form is over the counter oral tablets. The estimated average daily adult caffeine consumption is 300 mg. Rarely, serious toxicities such as seizure and cardiac arrhythmias have caused death. Caffeine concentrations of 80 mg/L are considered lethal. Cases investigated over 10 years by the Office of the Chief Medical Examiner for the State of Maryland (OCME) whose cause of death was solely or in combination due to caffeine intoxication were reviewed. A total of eight adult cases were identified consisting of two suicides and six undetermined manners. The average postmortem caffeine concentration was 140.4 mg/L. This is the largest case series reported to date of lethal caffeine intoxication. Large doses of prescription medications and/or over the counter medications were abused. No overdoses were from consumption of caffeine-containing foods. Comprehensive postmortem toxicological testing should test for caffeine to capture these rare deaths. [ABSTRACT FROM AUTHOR]

Abstract: Assigning a level of significance to cyanide concentrations found in the blood of fire victims is often hampered by the fact that cyanide is inherently unstable in cadavers and in stored blood samples. A few researchers have proposed that sodium fluoride can be used to minimize the instability of cyanide in blood samples; however, controlled studies have not been performed to support validation of this hypothesis. To test the sodium fluoride hypothesis, both treated and control blood samples from 14 autopsied fire victims were tested over a 25–30 day period. A 2% concentration of sodium fluoride was added to the blood samples at the start of testing and the samples were refrigerated between testing intervals. Cyanide concentrations in the treated and control samples were measured between 9 and 11 days post treatment and between 25 and 30 days post treatment. A statistically significant difference was not present between blood cyanide concentrations in treated and control samples between 9 and 11 days. During this time period, although there were small statistically significant increases in both treated and untreated samples the fluctuations were minor. Since the treated and control samples did not exhibit instability between 9 and 11 days, it is not surprising that the sodium fluoride appeared to have no effect. However, a statistically significant difference between blood cyanide concentrations in treated and control samples was observed between 25 and 30 days. Those samples treated with sodium fluoride showed a reduction in blood cyanide variability with virtually no overall change, over a 25–30 day period when compared to control samples, while unconditioned samples showed a significant, average increase of 35%. Based on the findings of this study, it is recommended that 2% sodium fluoride be added to blood samples obtained from fire victims to reduce cyanide instability due to bacteriological activity. [Copyright &y& Elsevier]

The article presents a case study wherein the combination of 4-methymethcathinone (mephedrone) and heroin caused the accidental death of a 22-year-old male. The autopsy revealed needle marks in the lower legs and ankles of the decedent and detected morphine in the blood and urine samples using gas chromatography-mass spectrometry (GC-MS). The medical examiner determined the death was caused by accidental multiple-drug toxicity.

The article presents a study regarding the cyanide toxicology on fire victims' blood, corpse and stored tissue. According to the researchers, the modification of cyanide in tissues and blood is based on the first concentration of the compound when the victim died and how long the substance stayed in the corpse, stored and preserved.

The article examines the presence of 1-(3-trifluoromethylphenyl)-piperazine (TFMPP) and 1-benzylpiperazine (BZP) in urine samples using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and gas chromatography-mass spectrometry (GC-MS). The BZP and TFMPP were detected through a full GC-MS scan of the seven U.S. service members' urine analysis specimen. Meanwhile, the LC-ESI-MS confirmed that in selected ion monitoring mode (SIM) yielded urinary concentrations ranging from 13.0 to 429.1 milligram/liter (mg/L) for BZP and 0.79 to 25.4 mg/L for TFMPP.

Abstract: Indole-3-carbinol (I-3-C) and 3,3′-diindolylmethane (DIM) have been shown to reduce the incidence and multiplicity of cancers in laboratory animal models. Based on the observation that I-3-C induced hepatocyte hypertrophy when administered orally for 13 weeks to rats, a treatment and recovery study was undertaken to test the hypothesis that the induction of hepatocyte hypertrophy and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses. Additionally, we directly compared the effects of I-3-C to those of its principle metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and 50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day) and then one-half of the animals were not treated for an additional 28 days. Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2, 2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28 days significantly increased liver weights and CYP enzyme activities. The effects in males were more pronounced and persistent after recovery than the effects in females. The increased organ weights returned to control values after treatment. Conversely, DIM did not alter liver weights and had no effect on CYP activities after the 28-day treatment. Some changes in CYP activities were measured after the DIM recovery period but the magnitudes of the changes were considered biologically insignificant. The results show that I-3-C, but not DIM, induces reversible adaptive responses in the liver. [Copyright &y& Elsevier]

Abstract: A direct, versatile method for the determination of ethyl glucuronide (EtG), a biomarker of ethanol consumption, in urine has been developed using reversed-phase liquid chromatography with pulsed electrochemical detection (PED). EtG and methyl glucuronide (MetG), which serves as an internal standard, are readily separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH allows for the detection of all glucuronides using PED at a gold working electrode. Upon optimization, EtG was found to have a limit of detection of 0.03μg/mL (7pmol; 50μL injection volume) and repeatability at the limit of quantitation of 1.7%R.S.D. (relative standard deviation). Solid-phase extraction (SPE) using an aminopropyl phase was used to remove interferents in urine samples prior to their analysis. Compound recovery following SPE was approximately 50±2%. The forensic utility of this method was further validated by the analysis of 29 post-mortem urine specimens, whose results agreed strongly with certified determinations. [Copyright &y& Elsevier]

Discusses the increase number of admissions and deaths due to oxycodone abuse and overdose in the U.S. Identification of the drug in therapeutic drug-testing procedures; Result of routine gas chromatographic analysis of bile or urine; Evidence of known metabolic pathways of oxycodone in other species.

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 mM. ¹H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1-10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+-lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA ≈ 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the ¹H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of stearic hindrance due to the extended conformation of the lecithin polar group. [ABSTRACT FROM AUTHOR]

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121,213–219]. It is now established that this N-terminal region of the A l-light chain interacts directly with the C-terminal region of actin in the acto-Sl (A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+ (CH3)3 protons of α-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11 — 15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1 (A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3A1a residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (<1.5 rim) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance spectroscopy has been used to study the cation(Mg2+ , Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the biding reaction. Complex formation with troponin I and the drug intercative conformational response to cation coordination and the binding of the drug and troponin I are discussed. [ABSTRACT FROM AUTHOR]

Using assignments of resonances in the 1H NMR spectrum of calmodulin obtained by the use of large tryptic fragments of the molecule [Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J. P., Dobrowolski, Z., and Drabikowski, W. (1984) Eur. J. Biochem. 138, 281–289], the spectral changes which occur on Ca2+ binding to calmodulin have been examined in detail. Ca2+ binding occurs in two stages: the first two Ca2+ ions bind at sites III and IV (numbered from the N terminus) and the second two Ca2+ ions bind at sites I and II. The high-affinity binding causes perturbations of residues in both halves of the molecule, whereas binding at the two N-terminal sites only affects sidechains in that half of the molecule. The effects of binding Cd2+ to the Ca2+-binding sites of calmodulin have also been studied by 1H NMR. The cation induces spectral changes which are very similar to those seen for Ca2+, but some important differences do exist. [ABSTRACT FROM AUTHOR]

Two tryptic fragments of the Ca2+-binding protein calmodulin have been studied by high-resolution 1H NMR. TR1C (residues 1 – 77) spans the first two domains of the protein and TR2C (residues 78 – 148) spans the second two domains. The spectra indicate that each of the two-domain peptide assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modeled after the two EF-Hand Ca2+-binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+ -binding protein. [ABSTRACT FROM AUTHOR]