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Introduction: Mucopolysaccharidosis (MPS) is an inherited metabolic disorder that is part of the lysosomal disorders; the main characteristic is the deficiency of lysosomal enzymes responsible for the degradation of glycosaminoglycans except for type II or Hunter syndrome, which is X-linked recessive inheritance. Neurological symptoms are Down syndrome, carpal tunnel, communicating hydrocephalus, spinal cord compression, and hearing loss. Neuroimaging plays a fundamental role in the diagnosis of central nervous system (CNS) involvement, as well as the assessment of complications such as hydrocephalus and spinal cord compression. Objective: To determine the characteristic findings of neuroimaging diagnostic aids in patients with MPS type II or Hunter's disease. Methodology: The recommendations of the Cochrane Collaboration [Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)] were followed. The bibliographic search was carried out in PubMed, Embase, Scopus, and ScienceDirect. Considering all the publications made up to 13th May 2018. Results and discussion: A total of 19 articles of the 723 results obtained were considered. Neuroradiological studies in patients with MPS type II still do not report clear and specific patterns of this pathology. Decreased contrast between the cortex and the underlying white matter has been reported; however, another study stated that these changes were secondary to demyelination or gliosis, manifestations that are not pathognomonic of MPS II. Ventricular dilatation is also a constant in patients with MPS II, which seems to be related to a cognitive disability; larger studies are still needed to confirm this association. Conclusion: Patients with MPS type II have a variety of abnormalities that are reflected in neuroradiological manifestations. The most significant findings of these patients included changes in gray and white matter signaling, cerebral atrophy, ventriculomegaly, increased perivascular space, increased subarachnoid space, and changes in the craniocervical junction. These characteristics must be strictly monitored both in the routine review and in the evaluation of responses to new therapeutic strategies.

Contexto: el manejo de los pacientes con acromegalia actualmente incluye cirugía, radioterapia y terapia farmacológica. Aunque la cirugía es el tratamiento de primera línea, un gran porcentaje de pacientes van a requerir manejo adicional con radioterapia y farmacoterapia. Objetivo: revisar las características farmacológicas y los estudios clínicos de pegvisomant. Metodología: se revisaron las bases de datos electrónicas como Medline y Pubmed con el término pegvisomant y se analizaron los estudios clínicos y los datos relevantes de este medicamento en el manejo de acromegalia. Resultados: los análogos de somatostatina constituyen el tratamiento farmacológico de primera línea indicado en las guías de práctica la de esta entidad, no obstante, estudios recientes han reportado niveles muy variables de control de la enfermedad. Para lograr un mejor control se introdujo el pegvisomant, un análogo de la hormona de crecimiento que se une al receptor pero no induce cambios conformacionales para la transducción de señales y, por tanto, no inhibe la secreción de la hormona de crecimiento, sino sus efectos fisiológicos en los tejidos diana. Hasta la fecha, ha demostrado eficacia para normalizar los niveles séricos del IGF-1 tanto en monoterapia como en terapia combinada. Conclusiones: si no logramos el control de la enfermedad a pesar de estos medicamentos, debemos considerar terapias de segunda línea como pasireotide o pegvisomant. Las características clínicas individuales del paciente permitirán elegir cuál es la mejor opción para nuestros pacientes. El pegvisomant es un medicamento eficaz para el control del paciente con acromegalia.

Abstract Intracerebral hemorrhage is hemorrhage occurring in the brain that can occur because of immediate trauma or structural disease process. The age-standardized mortality rate is decreasing worldwide especially in the past 20 years, but the number of cases and incidence along with number of disability-adjusted life-years and deaths keeps rising. Despite having half the incidence as ischemic stroke, intracerebral hemorrhage (traumatic or spontaneous) causes more morbidity and mortality than ischemic stroke and yet an established strategy for treatment on the lines of reperfusion therapy is a far-fetched dream. A selective review of all relevant articles covering recent treatment options and guidelines along with major emphasis of the surgical aspect and pathophysiology has been done. Complete literature review and differences between management of traumatic and spontaneous hemorrhage were sought and discussed. A pathophysiological basis of management should be followed for both these different yet similar pathologies. Understanding the etiology reveals a somewhat clearer picture regarding treatment of this highly unfortunate and devastating disease. Current interest is focused on the surgical management of the disease with the pathophysiology often been forgotten.

Spinal cord injury can lead to important functional, psychological and social sequelae. Despite the progress in medicine and greater understanding of the pathophysiological events associated with a traumatic spinal cord injury, spinal cord injury is still associated with a high morbidity and mortality. The involvement of the autonomic nervous system has implications in acute and chronic stages of the injured spinal cord patients. The most frequent cardiovascular complications in the acute phase of the traumatic spinal lesions are bradyarrhythmia, hypotension, increased vasovagal reflexes, ventricular and supraventricular ectopic beats, venous stasis, and vasodilation. In the chronic phase, we find orthostatic hypotension, alteration of the arterial pressure and the regulation of the body temperature as well as alteration of the blood volume. The knowledge of the cardiovascular alterations is of vital importance for the management and rehabilitation of the patients with spinal cord injury. In this article, we present a critical review of medical literature.

Introduction: Penetrating lesions by pencil in the temporal lobe in children and adolescents are uncommon. We present the case of a teenager with penetrating injury by strange object in the temporal lobe. Case: Twelve years old male patient, with history of trauma while he was playing with his friends, presents alteration of the consciousness state, weakness in right hemibody and dysphasia. Urgent surgery is practiced employing an incision in "C" form with improvement of the consciousness state during post-operative. Discussion: Penetrating lesions in the skull and brain are classified as missiles and non-missiles depending of their impact velocity. The wood is a porous organic material that provide a natural deposit of microbian agents, making it potentially lethal. Pre-operative radiological evaluation allows check the trajectory of the penetrating object and secondary lesions present guiding de neurosurgical approach. The prognostic depends on penetration site, timely handling and complications associated. Conclusion: Penetrating lesions by pencil are uncommon, an appropriate imaging evaluation is fundamental to determine the neurosurgical approach that allows prevent and/or decrease secondary damage.

Hydatidosis is a common disease worldwide. The causal agent may compromise any Organ of the body, the cerebral location is infrequent. The infection is caused by the larval form of Cestodo Echinococcus granulosus. The man is an accidental intermediary host for food consumption or water contaminated with eggs present in animal feces. We present a review of the literature. At the imaging level, the disease has a classic characteristic consisting of single, usually unilocular and less frequent multilocular, intra-axial and more frequently hemispheric cerebral lesions, compromising the vascular territory of the middle cerebral artery by the hematogenous dissemination of the parasite.

Oxidative stress constitute one of the commonest mechanism of the secondary injury contributing to neuronal death in traumatic brain injury cases. The oxidative stress induced secondary injury blockade may be considered as to be a good alternative to improve the outcome of traumatic brain injury (TBI) treatment. Due to absence of definitive therapy of traumatic brain injury has forced researcher to utilize unconventional therapies and its roles investigated in the improvement of management and outcome in recent year. Antioxidant therapies are proven effective in many preclinical studies and encouraging results and the role of antioxidant mediaction may act as further advancement in the traumatic brain injury management it may represent aonr of newer moadlaity in neurosurgical aramamentorium, this kind of therapy could be a good alternative or adjuct to the previously established neuroprotection agents in TBI.

Myelomeningocele may be associated with other neural and extraneural anomalies. Authors present association of metopic suture abnormality, an interfrontal encephalocele with widening of metopic suture and abnormal shape frontal bones in the forehead in those associated with hydrocephalus. Authors describes two neonates with interfrontal encephalocele, representing first series reporting in neonate. Management and pertinent literature is briefly discussed.

Acute traumatic brain injury is a worldwide public health crisis. Post-traumatic subarachnoid hemorrhage (SAH) is a finding that is present at a frequency of 40% according to data from American TCDB (1,2). Among the mechanisms that have been implicated as causes of post-traumatic SAH is the cortical bleeding through the subarachnoid space. It is estimated that the incidence of post-traumatic SAH is 11% to 60% in patients with traumatic brain injury. The brain CT is considered a technique with good sensitivity, economical and available in many hospitals worldwide. We have reviewed the literature and found some imaging characteristics of post-traumatic subarachnoid hemorrhage. We have called “the five preferences for post-traumatic SAH”.

Background. Conductive Education for stroke survivors has shown promise but randomised evidence is unavailable. This study assessed the feasibility of a definitive randomised controlled trial to evaluate efficacy. Methods. Adult stroke survivors were recruited through local community notices. Those completing the baseline assessment were randomised using an online program and group allocation was independent. Intervention group participants received 10 weekly 1.5-hour sessions of Conductive Education at the National Institute of Conductive Education in Birmingham, UK. The control group participants attended two group meetings. The study evaluated the feasibility of recruitment procedures, delivery of the intervention, retention of participants, and appropriateness of outcome measures and data collection methods. Independent assessments included the Barthel Index, the Stroke Impact Scale, the Timed Up and Go test, and the Hospital Anxiety and Depression Scale. Results. Eighty-two patients were enrolled; 77 completed the baseline assessment (46 men, mean age 62.1 yrs.) and were randomised. 70 commenced the intervention (n = 37) or an equivalent waiting period (n = 33). 32/37 completed the 10-week training and 32/33 the waiting period. There were no missing items from completed questionnaires and no adverse events. Discussion. Recruitment, intervention, and assessment methods worked well. Transport issues for intervention and assessment appointments require review. Conclusion. A definitive trial is feasible. This trial is registered with ISRCTN84064492.

Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1.

Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715-793) and one is in domain 4a (amino acids 636-714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a: H10 [622-708], H12 [506-708] and H13 [622-726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin-tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b.

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.

Interaction of S100a and S100b with duck gizzard caldesmon was investigated by means of native gel electrophoresis, fluorescent spectroscopy and disulfide crosslinking. Both isoforms of S100 interact with intact caldesmon and its C-terminal deletion mutant 606C (residues 606-756) with apparent Kd of 0.2-0.6 microM thus indicating that the S100-binding site is located in the C-terminal domain of caldesmon. The single SH group of duck gizzard caldesmon can be crosslinked to Cys-84 of the beta-chain or to Cys-85 of the alpha-chain of S100. Crosslinking of S100 reduces the inhibitory action of caldesmon on actomyosin ATPase activity. S100 reverses the inhibitory action of intact caldesmon and its deletion mutants 606C (residues 606-756) and H9 (residues 669-737) as effectively as calmodulin. S100a has higher affinity to caldesmon and is more effective than S100b in reversing caldesmon-induced inhibition of actomyosin ATPase activity. Although monomeric (calmodulin, troponin C) and dimeric (S100) Ca-binding proteins have different sizes and structures they interact with caldesmon in a very similar fashion.

Caldesmon interaction with smooth muscle myosin and its ability to cross-link actin filaments to myosin were investigated by the use of several bacterially expressed myosin-binding fragments of caldesmon. We have confirmed the presence of two functionally different myosin-binding sites located in domains 1 and 3/4a of caldesmon. The binding of the C-terminal site is highly sensitive to ionic strength and hardly participates in acto-myosin cross-linking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains two separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involved in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosin filaments, without affecting the affinity of direct acto-myosin interaction. Binding of caldesmon and its fragments to myosin or rod filaments revealed affinity in the micromolar range. We determined various stoichiometries at maximal binding, which depended on the ionic strength and the concentration of Mg2+ ions. At 30 mM NaCl and 1 mM Mg2+ the maximum stoichiometry was 4 moles of caldesmon (or caldesmon fragment) per mole of myosin. At 130 mM NaCl/1 mM Mg2+, or at 30 mM NaCl/5mM Mg2+ it decreased to about two caldesmon molecules bound per myosin, while remaining 4:1 for individual caldesmon fragments, suggesting that all binding sequences on myosin were still fully capable of interaction. A further increase in the Mg2+ concentration led to a substantial decrease in both the affinity and maximum stoichiometry of caldesmon and the fragments binding to myosin. We suggest that caldesmon-myosin interaction varies according to the conformation of caldesmon in solution, that caldesmon-binding sites on myosin are not well defined and that their accessibility is determined by spatial organization and is blocked by divalent cations like Mg2+.

The interaction of intact calmodulin and its four tryptic peptides with deletion mutants of caldesmon was analysed by native gel electrophoresis, fluorescence spectroscopy and zero-length cross-linking. Deletion mutants H2 (containing calmodulin-binding sites A and B) and H9 (containing sites B and B') interacted with intact calmodulin to form complexes whose stoichiometries varied from 2:1 to 1:1. The N-terminal peptides of calmodulin (TR1C, residues 1-77, and TR2E, residues 1-90) bound H2 with higher affinity than H9. At the same time H2 was less effective than H9 in binding to the C-terminal peptides of calmodulin TR2C (residues 78-148) and TR3E (residues 107-148). The N-terminal peptides of calmodulin (TR1C and TR2E) could be cross-linked to intact caldesmon and its deletion mutants H2 and H9. The similarity in the primary structures of sites A and B' of caldesmon and our measurements of the affinities of H2 and H9 to calmodulin and its peptides strongly indicate an orientation of the protein complex where sites A and B' interact with the N-terminal domain of calmodulin, whereas site B interacts with the C-terminal domain of calmodulin. The spatial organization of contact sites in the caldesmon-calmodulin complex agrees with the earlier proposed two-dimensional model of interaction of the two proteins [Huber, El-Mezgueldi, Grabarek, Slatter, Levine and Marston (1996) Biochem. J. 316, 413-420].

The interaction of caldesmon domains with tropomyosin has been studied using x-ray crystallography and an optical biosensor. Only whole caldesmon and the carboxyl-terminal domain of caldesmon (CaD-4, chicken gizzard residues 597-756) bound to tropomyosin with greater than millimolar affinity at 100 and 150 microM salt. Under these conditions the affinities of whole caldesmon and CaD-4 were both in the micromolar range. Data from the x-ray studies showed that whole caldesmon bound to tropomyosin in several places, with the region of tightest interaction being at tropomyosin residues 70-100 and/or 230-260. Studies with CaD-4 revealed that this region corresponded to the strong binding site seen with whole caldesmon. Weaker association of other regions of caldesmon to tropomyosin residues 180-210 and 5-50 was also observed. The results suggest that the carboxyl-terminus of caldesmon binds tightly to tropomyosin and that other regions of caldesmon may interact with tropomyosin tightly only when they are held close to tropomyosin by the carboxyl-terminal domain. Four models are presented to show the possible interactions of caldesmon with tropomyosin.

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.

We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding.

We have demonstrated that caldesmon does not alter the affinity of weak binding actomyosin complexes when it inhibits actin-tropomyosin activation at physiological ratios (1 per 14 actins), and we proposed that it acts upon the strong binding complexes in the same way that troponin-tropomyosin does. We therefore compared the effect of caldesmon, caldesmon fragments, and troponin upon the interaction of the strongly bound complexes S-1.ADP, S-1.adenylyl imidodiphosphate (AMP.PNP), and N-ethylmaleimide-treated myosin subfragment-1 (NEM-S-1) with actin-tropomyosin. In 0.17 M ionic strength buffer [14C]iodoacetamide-labeled S1.ADP bound to actin-smooth muscle tropomyosin with no evidence of cooperativity; Kd = 0.8 +/- 0.3 microM (n = 5). Inhibitory concentrations of sheep aorta caldesmon or rabbit skeletal muscle troponin made the binding highly cooperative. At low levels of saturation the apparent Kd was 10-40 microM with 10 microM caldesmon and 8-20 microM with 6 microM troponin; at > 50% saturation the binding was indistinguishable from actin-tropomyosin alone. A similar result was obtained for the binding of [14C]iodoacetamide-labeled S-1.AMP.PNP to actin-smooth muscle tropomyosin at 0.03 M ionic strength (Kd = 0.47 +/- 0.05 microM). Binding was slightly cooperative and became highly cooperative in the presence of inhibitory concentrations of troponin, caldesmon, and the human caldesmon fragments H7 (amino acids 622-767) and H9 (amino acids 726-793). We conclude that caldesmon and troponin both act as allosteric effectors of the "on"/"off" equilibrium of actin-tropomyosin. 0.1 NEM-S-1/actin potentiated actin-smooth muscle tropomyosin activation of myosin MgATPase 7-fold at 0.03 M ionic strength. Caldesmon inhibited the ATPase in the presence and absence of 0.5 microM NEM-S-1. NEM-S-1 reactivated actin-tropomyosin, which had been inhibited by troponin, caldesmon, H7, or H9. This is compatible with opposing effects of NEM-S-1 and caldesmon or troponin upon the actin-tropomyosin on/off equilibrium.

The presence of a silicone elastomer collar around one carotid artery of a rabbit induces thickening of the tunica intima. We used immunoblotting to study quantitatively changes in the isoforms of caldesmon, a protein implicated in the regulation of contractility in smooth muscle, while also monitoring the histological changes during 28 days after collaring. Control rabbit carotid arteries (n = 28) contained 245 +/- 6.4 nmol/g protein of the larger isoform of caldesmon (CDh) and 68.3 +/- 3.6 nmol/g protein of the smaller isoform (CD1). Four days after collaring, intimal thickening was slight, but 44% of arterial CDh had been lost; this loss of CDh was therefore from the tunica media. At 10 days, CDh fell to 37% of the control level. Immunofluorescence using CDh-specific antibodies showed that the CDh level was diminished but remained uniform across the wall of collared arteries. At 14 days, when intimal thickening was maximal, there was 30% more CD1 than in controls. At 28 days, the neointima had thinned, and CD1 had fallen to below control levels. Thus, CD1 levels reflected the development and regression of neointima. Changes in caldesmon isoforms showed that smooth muscle cell phenotypic changes occurred throughout the arterial wall.

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C-terminal part of caldesmon (so-called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606-756), H7 (566-710) and H2 (626-710) form tight complexes with phosphatidylserine, whereas fragments H8 (658-737), H9 (669-737) and fragment H4 (566-624) interact with phospholipids less effectively. It is concluded that the phospholipid-binding site is located in the sequence 626-710 of caldesmon. This sequence contains calmodulin-binding sites and serine residues phosphorylated by protein kinase C and pro-directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon-phospholipid interaction described earlier.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.