Venepuncture during head-up tilt testing in patients with suspected vasovagal syncope ­ implications for the test protocol.

BACKGROUND AND PURPOSE: Head-up tilt (HUT) testing is a widely used diagnostic tool in patients with suspected vasovagal syncope (VVS). However, no gold standard exists for this examination and the various protocols used have a limited sensitivity and specificity. Our aim was to determine the sensitivity of a sequential HUT testing protocol including venepuncture (VP) and sublingual nitroglycerin application. METHODS: This was a retrospective analysis of the diagnostic gain of a sequential HUT testing protocol including VP applied 10 min after the start of HUT testing and sublingual application of nitroglycerin 20 min after the start of the test protocol in 106 patients with a final diagnosis of VVS. The sensitivity of the test protocol was compared between patients with positive and negative history for VP induced VVS. RESULTS: Overall, pre-syncope or syncope occurred in 68 patients (64.2%). Only 17% of all patients fainted spontaneously within 10 min of passive HUT. Another 39.6% fainted within 20 min. Application of nitroglycerin after 20 min of HUT evoked syncope in another 7.5% until the end of 45 min of HUT. The sensitivity of the test protocol for evoking (pre-)syncope was 94.4% in patients with a positive history for VP associated VVS and 58% in patients with a negative history (P < 0.01**); 85.7% of patients with a positive history and 42.9% of patients with a negative history fainted within 20 min of HUT testing (P < 0.01**). CONCLUSIONS: Implementation of VP in sequential HUT testing protocols allows the sensitivity of HUT testing to be increased, especially in patients with a positive history for VP associated VVS.

[Orthostatic headache with tachycardia]. / Orthostatische Kopfschmerzen und Tachykardie.

We report the case of a 17 year old male patient who presented with a history of orthostatic headache (present in the upright position only) for several months. The diagnostic investigations (MRI of the head and of the spine, lumbar puncture) revealed no signs of an intracranial hypotension or a CSF leak. In standing position, a significant raise of the heart rate (>40 bpm) without fall of the blood pressure occurred together with a bilateral, pressure-like headache. A diagnosis of postural tachycardia syndrome was made. Treatment with increase of fluid and salt intake, elastic compression stockings and regular exercise was successful.

Abnormal cardiovascular responses to carotid sinus massage also occur in vasovagal syncope - implications for diagnosis and treatment.

BACKGROUND AND PURPOSE: Carotid sinus massage (CSM) is commonly used to identify carotid sinus hypersensitivity (CSH) as a possible cause for syncope, especially in older patients. However, CSM itself could provoke classical vasovagal syncope (VVS) in pre disposed subjects. METHODS: Retrospective analysis of CSM, cardiovascular autonomic function tests (including tilt table testing) and medical history in 388 patients with recurrent syncope to identify and characterize patients in whom an abnormal response to CSM was more likely to reflect VVS than CSH. RESULTS: CSM was abnormal in 79 patients. In 53 patients (77.2 +/- 8.7 years), CSH was the likely cause of syncope. VVS was the more likely diagnosis in 26 younger patients (59.7 +/- 12.6 years) with longstanding syncope from youth, in whom fear or pain was as a trigger; 7/26 suffered from intense chronic or intermittent neck pain and one exacerbation of syncopal attacks followed a physical and emotional trauma to the neck. In VVS, 4/26 had spontaneous VVS during head-up tilt, another six after venepuncture (performed in 17/26). In 6/26, the abnormal response to CSM was delayed, occurring 62.8 +/- 28.4 s after completion of CSM. The response to CSM was predominantly of the mixed type (20/26) and abnormal on both sides in 14/26. CONCLUSIONS: An abnormal response to CSM may not indicate syncope caused by CSH and needs to be considered in the light of the patient's age, duration of syncopal episodes and detailed history of provocative stimuli. Differentiating CSH from VVS with an abnormal response to CSM has various implications from advice on driving to treatment strategies.

Acute fluid ingestion in the treatment of orthostatic intolerance - important implications for daily practice.

BACKGROUND: Rapid water ingestion improves orthostatic intolerance (OI) in multiple system atrophy (MSA) and postural tachycardia syndrome (PoTS). We compared haemodynamic changes after water and clear soup intake, the latter being a common treatment strategy for OI in daily practice. METHODS: Seven MSA and seven PoTS patients underwent head-up tilt (HUT) without fluid intake and 30 min after drinking 450 ml of water and clear soup, respectively. All patients suffered from moderate to severe OI because of neurogenic orthostatic hypotension (OH) and excessive orthostatic heart rate (HR) increase, respectively. Beat-to-beat cardiovascular indices were measured non-invasively. RESULTS: In MSA, HUT had to be terminated prematurely in 2/7 patients after water, but in 6/7 after clear soup. At 3 min of HUT, there was an increase in blood pressure of 15.7(8.2)/8.3(2.3) mmHg after water, but a decrease of 11.6(18.9)/8.1(9.2) mmHg after clear soup (P < 0.05). In PoTS, HUT could always be completed for 10 min, but OI subjectively improved after both water and clear soup. The attenuation of excessive orthostatic HR increase did not differ significantly after water and clear soup drinking. CONCLUSIONS: In MSA, clear soup cannot substitute water for eliciting a pressor effect, but even worsens OI after rapid ingestion. In PoTS, acute water and clear soup intake both result in improvement of OI. These findings cannot solely be explained by difference in osmolarity but may reflect some degree of superimposed postprandial hypotension in widespread autonomic failure in MSA compared to the mild and limited autonomic dysfunction in PoTS.

Velocity recovery cycles of human muscle action potentials in chronic renal failure.

OBJECTIVE: To test the hypothesis that muscle fibers are depolarized in patients with chronic renal failure, by measuring velocity recovery cycles of muscle action potentials as indicators of muscle membrane potential. METHODS: Velocity recovery cycles were recorded from brachioradialis muscle by direct muscle stimulation in 13 patients, before, immediately after, and 1h after haemodialysis, and compared with those from 10 age-matched controls. RESULTS: In the patients, supernormality was reduced by 47%, and relative refractory period increased by 60.5% compared with controls (both P<0.001). Dialysis normalized the supernormality, but an hour later it was again reduced. These changes in supernormality were strongly correlated with the changes in serum potassium levels (P<0.0001). A late component of supernormality, attributed to potassium accumulation in the t-tubule system, was also reduced in the patients but remained abnormally low after dialysis. CONCLUSIONS: Muscle membranes in the patients were chronically depolarized by hyperkalemia. Whereas dialysis transiently normalized muscle membrane potential, it was not adequate to normalize t-tubule function. SIGNIFICANCE: Chronic muscle membrane depolarization by hyperkalemia may account for some of the functional deficits in uremic myopathy. Consistent normalization of membrane potential by avoiding hyperkalemia may therefore reduce symptoms of 'uremic myopathy'.

Repetitive spinal motor neuron discharges following single transcranial magnetic stimulation: relation to dexterity.

Transcranial magnetic stimulation allows to study the properties of the human corticospinal tract non-invasively. After a single transcranial magnetic stimulus, spinal motor neurons (MNs) sometimes fire not just once, but repetitively. The biological significance of such repetitive MN discharges (repMNDs) is unknown. To study the relation of repMNDs to other measures of cortico-muscular excitability and to physiological measures of the skill for finely tuned precision movements, we used a previously described quadruple stimulation (QuadS) technique (Z'Graggen et al. 2005) to quantify the amount of repMNDs in abductor digiti minimi muscles (ADMs) on both sides of 20 right-handed healthy subjects. Skillfulness for finger precision movements of both hands was assessed using a finger tapping task. In 16 subjects, a follow-up examination was performed after training of either precision movements (n = 8) or force (n = 8) of the left ADM. The size of the QuadS response (amplitude and area ratios) was greater in the dominant right hand than in the left hand (QuadS amplitude ratio: 47.1 +/- 18.1 versus 37.7 +/- 22.0%, Wilcoxon test: P < 0.05; QuadS area ratio: 49.7 +/- 16.2% versus 36.9 +/- 23.0%, Wilcoxon test: P < 0.05), pointing to a greater amount of repMNDs. Moreover, the QuadS amplitude and area increased significantly after finger precision training, but not after force training. This increase of repMNDs correlated significantly with the increase in performance in the finger tapping task. Our results demonstrate that repMNDs are related to handedness and therefore probably reflect supraspinal excitability differences. The increase of repMNDs after skills training but not after force training supports the hypothesis of a supraspinal origin of repMNDs.

Effects of water drinking on cardiovascular responses to supine exercise and on orthostatic hypotension after exercise in pure autonomic failure.

OBJECTIVE: Patients with pure autonomic failure (PAF) have an abnormal fall in blood pressure (BP) with supine exercise and exacerbation of orthostatic hypotension (OH) after exercise. This study assessed the pressor effect of water on the cardiovascular responses to supine exercise and on OH after exercise. METHODS: 8 patients with PAF underwent a test protocol consisting of standing for 5 min, supine rest for 10 min, supine exercise by pedalling a cycle ergometer at workloads of 25, 50 and 75 W (each for 3 min), supine rest for 10 min and standing for 5 min. The test protocol was performed without water ingestion and on a separate occasion after 480 ml of distilled water immediately after pre-exercise standing. Beat to beat cardiovascular indices were measured with the Portapres II device with subsequent Modelflow analysis. RESULTS: All patients had severe OH pre-exercise (BP fall systolic 65.0 (26.1) mm Hg, diastolic 22.7 (13.5) mm Hg), with prompt recovery of BP in the supine position. 5 min after water drinking, there was a significant rise in BP in the supine position. With exercise, there was a clear fall in BP (systolic 42.1 (24.4) mm Hg, diastolic 25.9 (10.0) mm Hg) with a modest rise in heart rate; this occurred even after water ingestion (BP fall systolic 49.8 (18.9) mm Hg, diastolic 26.0 (9.1) mm Hg). BP remained low after exercise but was significantly higher after water intake, resulting in better tolerance of post-exercise standing. CONCLUSIONS: Water drinking did not change the abnormal cardiovascular responses to supine exercise. However, water drinking improved orthostatic tolerance post-exercise.

Unexplained syncope--is screening for carotid sinus hypersensitivity indicated in all patients aged >40 years?

OBJECTIVE: To determine the frequency, age distribution and clinical presentation of carotid sinus hypersensitivity (CSH) among 373 patients (age range 15-92 years) referred to two autonomic referral centres during a 10-year period. METHODS: Carotid sinus massage (CSM) was performed both supine and during 60 degree head-up tilt. Beat-to-beat blood pressure, heart rate and a three-lead electrocardiography were recorded continuously. CSH was classified as cardioinhibitory (asystole > or = 3 s), vasodepressor (systolic blood pressure fall > or = 50 mm Hg) or mixed. All patients additionally underwent autonomic screening tests for orthostatic hypotension and autonomic failure. RESULTS: CSH was observed in 13.7% of all patients. The diagnostic yield of CSM was nil in patients aged < 50 years (n = 65), 2.4% in those aged 50-59 years (n = 82), 9.1% in those aged 60-69 years (n = 77), 20.7% in those aged 70-79 years (n = 92) and reached 40.4% in those > 80 years (n = 57). Syncope was the leading clinical symptom in 62.8%. In 27.4% of patients falls without definite loss of consciousness was the main clinical symptom. Mild and mainly systolic orthostatic hypotension was recorded in 17.6%; evidence of sympathetic or parasympathetic dysfunction was found in none. CONCLUSIONS: CSH was confirmed in patients > 50 years, the incidence steeply increasing with age. The current European Society of Cardiology guidelines that recommend testing for CSH in all patients > 40 years with syncope of unknown aetiology may need reconsideration. Orthostatic hypotension was noted in some patients with CSH, but evidence of sympathetic or parasympathetic failure was not found in any of them.

Quantification of central motor conduction deficits in multiple sclerosis patients before and after treatment of acute exacerbation by methylprednisolone.

OBJECTIVE: To compare the effects of intravenous methylprednisolone (IVMP) in patients with relapsing-remitting (RR-MS), secondary progressive (SP-MS), and primary progressive multiple sclerosis (PP-MS). METHODS: Clinical and neurophysiological follow up was undertaken in 24 RR-MS, eight SP-MS, and nine PP-MS patients receiving Solu-Medrol 500 mg/d over five days for exacerbations involving the motor system. Motor evoked potentials (MEPs) were used to measure central motor conduction time (CMCT) and the triple stimulation technique (TST) was applied to assess conduction deficits. The TST allows accurate quantification of the number of conducting central motor neurones, expressed by the TST amplitude ratio. RESULTS: There was a significant increase in TST amplitude ratio in RR-MS (p<0.001) and SP-MS patients (p<0.02) at day 5, paralleling an increase in muscle force. TST amplitude ratio and muscle force remained stable at two months. In PP-MS, TST amplitude ratio and muscle force did not change. CMCT did not change significantly in any of the three groups. CONCLUSIONS: In RR-MS and SP-MS, IVMP is followed by a prompt increase in conducting central motor neurones paralleled by improvement in muscle force, which most probably reflects partial resolution of central conduction block. The lack of similar clinical and neurophysiological changes in PP-MS corroborates previous clinical reports on limited IVMP efficacy in this patient group and points to pathophysiological differences underlying exacerbations in PP-MS.

Repetitive spinal motor neuron discharges following single transcranial magnetic stimuli: a quantitative study.

OBJECTIVE: To quantify repetitive discharges of spinal motor neurons (repMNDs) in response to single transcranial magnetic stimuli (TMS). To assess their contribution to the size of motor evoked potentials (MEPs). METHODS: We combined the triple stimulation technique (TST) with an additional nerve stimulus in the periphery (= quadruple stimulation; QuadS). The QuadS eliminates the first action potential descending on each axon after TMS, and eliminates effects on response size induced by desynchronization of these discharges, thereby allowing a quantification of motor neurons (MNs) discharging twice. In some instances, a quintuple stimulation (QuintS) was used, to quantify the number of MNs discharging three times. Recordings were from the abductor digiti minimi of 14 healthy subjects, using two different stimulation intensities and three different levels of facilitatory muscle pre-contractions. RESULTS: The threshold to obtain repMNDs was high. Their maximal size differed markedly between subjects, ranging from 8 to 52% of all MNs. Stimulation intensity and facilitatory muscle contraction, but not resting motor threshold, correlated with the amount of repMNDs. QuintS never yielded discernible responses, hence all observed repMNDs were double discharges. RepMNDs contributed to the MEP areas, but did not influence MEP amplitudes. CONCLUSIONS: QuadS and QuintS allow precise quantification of repMNDs. The threshold of repMNDs is high and varies considerably between subjects. SIGNIFICANCE: repMNDs have to be considered when MEP areas are measured. Their analysis may be of interest in neurological disorders, but standardized stimulation parameters appear essential.

Quantification of Uhthoff's phenomenon in multiple sclerosis: a magnetic stimulation study.

OBJECTIVE: To quantify temperature induced changes (=Uhthoff phenomenon) in central motor conduction and their relation to clinical motor deficits in 20 multiple sclerosis (MS) patients. METHODS: Self-assessment of vulnerability to temperature and clinical examination were performed. We used motor evoked potentials to measure central motor conduction time (CMCT) and applied the triple stimulation technique (TST) to assess conduction failure. The TST allows an accurate quantification of the proportion of conducting central motor neurons, expressed by the TST amplitude ratio (TST-AR). RESULTS: Temperature induced changes of TST-AR were significantly more marked in patients with prolonged CMCT (P=0.037). There was a significant linear correlation between changes of TST-AR and walking velocity (P=0.0002). Relationships were found between pronounced subjective vulnerability to temperature and (i) abnormal CMCT (P=0.02), (ii) temperature induced changes in TST-AR (P=0.04) and (iii) temperature induced changes in walking velocity (P=0.04). CMCT remained virtually unchanged by temperature modification. CONCLUSIONS: Uhthoff phenomena in the motor system are due to varying degrees of conduction block and associated with prolonged CMCT. In contrast to conduction block, CMCT is not importantly affected by temperature. SIGNIFICANCE: This is the first study quantifying the Uhthoff phenomenon in the pyramidal tract of MS patients. The results suggest that patients with central conduction slowing are particularly vulnerable to develop temperature-dependent central motor conduction blocks.

Assessment of central motor conduction to intrinsic hand muscles using the triple stimulation technique: normal values and repeatability.

OBJECTIVE: To establish the triple stimulation technique (TST) for recordings from the first dorsal interosseus (FDI) and the abductor pollicis brevis muscles (APB), and to analyse the test-retest repeatability of the TST measurements in APB. METHODS: The recently developed TST was slightly modified for recordings from small hand muscles to account for volume conducted activity from surrounding muscles. The TST combines transcranial magnetic stimulation (TMS) with a peripheral collision technique [Magistris et al. Brain 121 (1998) 437]. In contrast to conventional motor-evoked potentials (MEPs), it quantifies the number of conducting central motor neurons (expressed by the TST amplitude ratio, TST-AR). MEPs and TST were performed in 30 sides of 25 healthy subjects (target muscle FDI), and in 29 sides of 21 healthy subjects (target muscle APB). All APB recordings were repeated after 25+/-5.9 days. RESULTS: The TST-AR averaged 97.4+/-2.5% in FDI and 95.9+/-4.7% in APB. There was a mean difference of the TST-AR ratio of 2.9+/-3.1% between the repeated APB recordings (95% limits of agreement+/-6.3%). CONCLUSIONS: TMS allows activation of virtually all motor neurons supplying FDI and APB, when effects of volume conduction are eliminated. Its test-retest repeatability is excellent. SIGNIFICANCE: The TST is well suited for follow-up examinations of central motor conduction failures. The greater number of established target muscles widens its clinical applicability.

Central motor conduction differs between acute relapsing-remitting and chronic progressive multiple sclerosis.

OBJECTIVE: To characterize central motor conduction in relation to the clinical deficits and to the disease duration in 90 patients with acute relapsing-remitting MS (RR-MS) and in 51 patients with chronic primary or secondary progressive MS (P-MS). METHODS: The triple stimulation technique (TST) was used to quantify the central motor conduction failure (expressed by the TST amplitude ratio) and conventional motor evoked potentials (MEPs) were used to measure the central motor conduction time (CMCT). RESULTS: The TST amplitude ratio was reduced in presence of a clinical motor deficit (p=0.02 for RR-MS, p<0.01 for P-MS), but did not significantly differ in RR-MS and P-MS (p>0.05) when patients with similar clinical motor deficit were compared. The CMCT was not related to the clinical motor deficit in both RR-MS and P-MS. However, the CMCT was markedly prolonged in P-MS, when patients with similar clinical motor deficit and with similar disease duration were compared (p<0.01). The differences were not attributable to differential involvement of the spinal cord, which was similar in RR-MS and P-MS. CONCLUSIONS: Our results disclose differences between the central motor conduction in RR-MS and P-MS that are not related to disease severity, spinal cord involvement or disease duration.

Abnormal trafficking of sarcolemmal proteins in alpha-glucosidase deficiency.

The dystrophin-associated protein complex (DAP) plays an important role in the integrity and stability of the muscle membrane. Whereas much is known about the interaction between DAP members at the sarcolemmal location, intracellular DAP assembly and trafficking is still largely unknown. In alpha-glucosidase (acid maltase) deficiency (alphaGDD), accumulation of glycogen is accompanied by cytoarchitectural abnormalities impairing normal protein metabolism. In the present study, we took advantage of this fact to examine the consequences of impaired protein handling on the formation of DAP, with the aim of gaining indirect knowledge about its sarcoplasmic trafficking and a better understanding of mechanisms leading to myopathic changes found in alphaGDD. Histological examination of alphaGDD muscle confirmed a vacuolar myopathy with glycogen accumulation both in vacuoles and within the sarcoplasm. Sarcoplasmic accumulation of sarcolemmal proteins, including dystrophin and sarcoglycans, occurred around some vacuoles and within non-vacuolated fibres. Utrophin was up-regulated and found at extra-junctional sarcolemmal locations of many fibres. AlphaGDD muscle cells developed in a fashion similar to that of controls in culture. However, vacuoles were found in 2-week-old alphaGDD myotubes, and these subsequently increased in size and number. Substantial alterations in DAP handling were found, with accumulation close to the Golgi apparatus. Utrophin was not enriched in the sarcoplasm but was up-regulated along the whole sarcolemma. Our results demonstrate a close association of dystrophin and sarcoglycans during sarcoplasmic processing. Furthermore, they suggest that the myopathy found in alphaGDD is a secondary form of DAP deficiency.

Women's emotions and concerns during pregnancy following perinatal loss.

PURPOSE: To determine the specific emotions and concerns of women who are pregnant following a perinatal loss. STUDY DESIGN AND METHODS: Data were collected through a mailed questionnaire using an open response format. The sample consisted of 73 women on the membership mailing lists from two pregnancy-after-loss support groups. A content analysis was conducted on women's self-reports; five primary emotions and five main concerns were expressed during their pregnancies. RESULTS: "Anxious," "nervous," and "scared" were the most frequent emotions reported by these women. However, most women also included a positive emotion in their list, indicating the mixed emotions of their pregnancy experience. Eight categories of profound concerns were identified: "losing another baby," "overall health of the baby," "emotional stability of self," "impact of another loss on my future," "lack of support from others," "fear of bad news," "own impact on the baby," and "worries never end." Responses of currently pregnant women as compared with women retrospectively reporting on their last pregnancy experiences were essentially similar. CLINICAL IMPLICATIONS: Women pregnant after a previous perinatal loss are skeptical about pregnancy. Clinicians should be cognizant of the constellation of concerns and the simultaneous contrasting emotions experienced by these women in order to provide supportive prenatal care. Because the women's concerns are ongoing, responsive care should include asking about specific concerns throughout the pregnancy.

Enkephalin and aFGF are differentially regulated in rat spinal motoneurons after chemodenervation with botulinum toxin.

Botulinum toxin is used to induce transient graded paresis by chemodenervation in the treatment of focal hyperkinetic movement disorders. While the molecular events occurring in motoneurons after mechanical nerve lesioning leading to muscle paresis are well known, they have been investigated to a lesser extent after chemodenervation. We therefore examined the expression of enkephalin (ENK), acidic fibroblast growth factor (aFGF), neurotensin (NT), galanin (GAL), substance P (SP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in rat spinal motoneurons after chemodenervation of the gastrocnemius. In order to precisely localize the motoneurons targeting the injection site, retrograde tracing was performed in additional rats by using Fluorogold injections. ENK expression was upregulated in the region corresponding to the Fluorogold positive motoneurons, but also on the contralateral side and in more distant parts of the spinal cord. The highest upregulation occurred 7 to 14 days after injections and decreased over a period of three months. At 8 days, aFGF was slightly downregulated in all regions studied, single motoneurons showed NT expression, while expression of GAL, SP, VIP, and NPY could be detected neither in controls nor in toxin-treated animals. These alterations in gene expression were strikingly different from those described after axotomy. Our present findings give additional demonstration of the considerable plasticity of the adult spinal cord after botulinum toxin treatment.

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.

BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

The ligand-induced structural changes of human L-Arginine:Glycine amidinotransferase. A mutational and crystallographic study.

Human L-arginine:glycine amidinotransferase (AT) shows large structural changes of the 300-flap and of helix H9 upon binding of L-arginine and L-ornithine, described as a closed and an open conformation (Humm, A., Fritsche, E., Steinbacher, S., and Huber, R. (1997) EMBO J. 16, 3373-3385). To elucidate the structural basis of these induced-fit movements, the x-ray structures of AT in complex with the amidino acceptor glycine and its analogs gamma-aminobutyric acid and delta-aminovaleric acid, as well as in complex with the amidino donor analogs L-alanine, L-alpha-aminobutyric acid, and L-norvaline, have been solved at 2.6-, 2.5-, 2.37-, 2.3-, 2.5-, and 2.4-A resolutions, respectively. The latter three compounds were found to stabilize the open conformer. The glycine analogs bind in a distinct manner and do not induce the transition to the open state. The complex with glycine revealed a third binding mode, reflecting the rather broad substrate specificity of AT. These findings identified a role for the alpha-amino group of the ligand in stabilizing the open conformer. The kinetic, structural, and thermodynamic properties of the mutants ATDeltaM302 and ATDelta11 (lacks 11 residues of H9) confirmed the key role of Asn300 and suggest that in mammalian amidinotransferases, the role of helix H9 is in accelerating amidino transfer by an induced-fit mechanism. Helix H9 does not add to the stability of the protein.

Crystal structure of L-arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus: an enzyme involved in streptomycin biosynthesis.

Inosamine-phosphate amidinotransferases catalyze two nonconsecutive transamidination reactions in the biosynthesis of the streptomycin family of antibiotics. L-Arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus (StrB1) was cloned as an N-terminal hexa-histidine fusion protein, purified by affinity chromatography, and crystallized, and its crystal structure was solved by Patterson search methods at 3.1 A resolution. The structure is composed of five betabeta alphabeta-modules which are arranged circularly into a pseudo-5-fold symmetric particle. The three-dimensional structure is closely related to the structure of human L-arginine:glycine amidinotransferase (AT), but five loops (the 40-, 170-, 220-, 250-, and 270-loop) are organized very differently. The major changes are found in loops around the active site which open the narrow active site channel of AT to form an open and solvent-exposed cavity. In particular, module II of StrB1 is AT-like but lacks a 10-residue alpha-helix in the 170-loop. The concomitant reorganization of neighboring surface loops that surround the active site, i.e., the 40-loop and the 270-loop, results in an arrangement of loops which allows an unrestricted access of substrates to the cavity. However, the residues which are involved in substrate binding and catalysis are conserved in AT and StrB1 and are at equivalent topological positions, suggesting a similar reaction mechanism among amidinotransferases. The binding site for L-arginine had been deduced from its complex with AT. Molecular modeling revealed a possible binding mode for the second substrate scyllo-inosamine 4-phosphate.