Biomedical subjects

A S Mak

Publications and source records attributed to A S Mak.

At least 19 recordsLinked to original sources

PubMed2000

Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction.

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.

Actins↗

PubMed2000

MAPK and PKC activity are not required for H(2)O(2)-induced arterial muscle contraction.

H(2)O(2)-induced pulmonary arterial smooth muscle (PASM) contractions are independent of Ca(2+) and myosin light chain phosphorylation. The purpose of this study was to determine whether mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1 and ERK2, or protein kinase C (PKC) activation is required for H(2)O(2)-induced contraction. Porcine PASM strips were stimulated with 1 mM H(2)O(2), 120 mM KCl, or 10 microM phorbol myristic acetate and freeze clamped at various times during the contractions. Changes in relative amounts of tyrosine/threonine phosphorylated MAPK compared with total MAPK were measured. MAPK tyrosine phosphorylation levels increased in correlation with tension development. However, 50 microM PD-98059, a MAPK/ERK kinase-MAPK kinase blocker, reduced MAPK phosphorylation below resting levels, even though the magnitude of the isometric tension development was unaltered. Freeze-clamped PASM strips were placed in a PKC activity assay buffer containing (32)P and CaCl(2) to measure the total myelin basic protein phosphorylation. The data show that: 1) the time courses of PKC activity and force produced in response to H(2)O(2) do not correlate, and 2) MAPK activation may be a concurrent event with, or a consequence of, tension development in response to a variety of agonists but is not responsible for contractions to H(2)O(2), high K(+), or phorbol esters.

Animals↗

PubMed1998

Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle.

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

Androstadienes↗

PubMed1997

NMR studies of caldesmon-calmodulin interactions.

The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the binding of one of these domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-1171). Here we have studied the binding of CaM to synthetic peptides of CaD which contain: (1) both the first and second CaM-binding domains; (2) the second CaM-binding domain; and (3) the sequence between the first and second CaM-binding domains. Two-dimensional transferred nuclear Overhauser enhancement proton NMR measurements as well as circular dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, which contains the second CaM-binding domain, show that only the C-terminal half of the peptide becomes alpha-helical upon binding to CaM. Somewhat surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10)-helix. Proton-carbon-13 correlation NMR titration studies with specifically labeled [methyl-13C]methionine CaM were used to study the participation of the hydrophobic regions in both domains of the dumbbell shaped CaM in peptide binding. Binding of a 54-residue CaD peptide containing both CaM-binding domains affects all the 8 Met residues in the two hydrophobic domains of CaM (only Met 76 in the linker region of CaM is not involved), while binding of the second CaM-binding domain of CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous binding to CaM of two peptides comprising the first and the second CaM-binding domains also caused changes to all Met residues except Met 76. Taken together, these data demonstrate that both CaM-binding domains of CaD can bind simultaneously to the two hydrophobic regions of CaM.

Amino Acid Sequence↗

PubMed1997

Tryptophan residues in caldesmon are major determinants for calmodulin binding.

Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 296, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a Kd of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31-fold (Kd = 2.9 x 10(-6) and 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol-1, respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol-1. On the other hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (Kd = 0.6 x 10(-6) M) and results in a destabilization energy of 0.87 kcal mol-1. To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A + B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (Kd = 1.7 x 10(-6) M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (Kd = 4.1 x 10(-6) M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmon-calmodulin interaction and that Trp 722 in site B' plays a minor role.

Amino Acid Sequence↗

PubMed1997

Autoradiographic localization of 2[125I]iodomelatonin binding sites in the gastrointestinal tract of mammals including humans and birds.

In-vitro autoradiography was utilized to compare the distribution of 2[125I]iodomelatonin binding sites or putative melatonin receptors in the gastrointestinal tracts of humans, guinea pigs, mice, rats, hamsters, rabbits, ducks, chickens, pigeons, and quail. In humans, binding was detected in the mucosa of the colon, caecum, appendix, and on their blood vessels but not in the ileum. In the other mammals, significant binding was only demonstrated in the mucosa of the rabbit rectum, mouse colon, mouse rectum, and guinea pig ileum. The distribution of 2[125I]iodomelatonin binding in the avian gut varied with species. In the esophagus, binding was present in the lamina propria and blood vessels of all four birds. However, only the lamina propria of the chicken and quail proventriculus and ventriculus showed positive binding. For the duodenum and ileum, binding was very strong in the duck lamina propria, weak in the chicken lamina propria, and absent in the quail. In contrast, the pigeon muscle layer was weakly positive. The most striking species difference was found in the caecum where the duck lamina propria showed very strong binding, while the chicken lamina propria was only weakly positive. Conversely, the caecal muscle layer was strongly positive in chicken and quail but negative in duck and pigeon. In the rectum, a similar but less intense pattern of distribution was observed. The tremendous diversity in the distribution of 2[125I]iodomelatonin binding sites in the gastrointestinal tract is in accord with the hypothesis that melatonin may serve different functions in the gut of different species.

Animals↗

PubMed1997

Skilled Hong Kong immigrants' intention to repatriate.

"An emphasis on skills in Australian immigration policy in the past decade has led to the increase of highly skilled Hong Kong immigrants. However, Australia has not been able to retain all of them.... This paper reports the results of an in-depth study on intention to repatriate and work in Hong Kong, conducted in Australia with 111 professional and managerial Hong Kong immigrants. Correlational and loglinear analyses on prediction of such an intention are presented. Research findings on the career-family dilemma experienced by a number of immigrants are likewise discussed."

Asia↗

PubMed1996

Identification of C-type natriuretic peptide gene transcripts in glial cells.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is found throughout the central nervous system (CNS), particularly in those regions involved in neuroendocrine regulation. Astrocytes, which have important physiological roles in normal neuronal functioning, express receptors of CNP. Using reverse transcription-polymerase chain reaction (RT-PCR), followed by hybridization with a digoxigenin-labelled cDNA probe, we have demonstrated the expression of CNP gene transcripts in both cultured mouse astrocytes and rat C6 glioma cells, with the former expressing the gene at a considerably higher level than the latter. Our data raise the possibility that CNP may act in autocrine and/or paracrine fashion in glial cell physiology and neuromodulate communication between glial cells and neurones.

Animals↗

PubMed1996

Melatonin and 2[125I]iodomelatonin binding sites in the human colon.

2[125I]Iodomelatonin binding sites were identified in the mucosa of the human colon from Chinese patients with carcinoma of the rectum or colon using biochemical receptor assay and autoradiography. Melatonin in the colonic mucosa/submucosa and muscle layers were quantitated by radioimmunoassay. The binding of 2[125I]iodomelatonin to the membrane preparations of the human colonic mucosa/submucosa was stable, saturable, reversible and of high affinity. Rosenthal analysis from saturation studies performed at 21 degrees C yielded an equilibrium dissociation constant (Kd) of 61.7 +/- 4.48 pmol/L (n = 3) and maximum number of binding sites (B(max)) of 1.65 +/- 0.51 fmol/mg protein (n = 3). The linearity of the Rosenthal plots and unity of the Hill coefficient suggested that 2[125I]iodomelatonin was bound to a single class of binding sites. The radioligand binding was displaced by 2-iodomelatonin (Ki = 0.02 nmol/L), melatonin (0.65 nmol/L), 6-chloromelatonin (Ki = 5.33 nmol/L), 6-hydroxymelatonin (Ki = 33.8 nmol/L) and N-acetylserotonin (Ki = 122 nmol/L). The characteristic of the binding sites were similar to those reported in the jejunum of duck, chicken, and human but of higher affinity than those in the mouse colon. Autoradiography localizes the binding to the mucosa of the human colon. Radioimmunoassay revealed a melatonin concentration of 467 +/- 99 pg/g wet tissue of human colon (n = 6). Our findings suggest that melatonin may influence the human colonic functions through interaction with its receptors in the mucosa.

Aged↗

PubMed1995

Alignment of caldesmon on the actin-tropomyosin filaments.

We have reported previously that each smooth-muscle caldesmon binds predominantly to a region within residues 142-227 of tropomyosin, but a weaker binding site also exists at the N-terminal region of tropomyosin [Watson, Kuhn, Novy, Lin and Mak (1990) J. Biol. Chem. 265, 18860-18866]. In view of recent evidence for the presence of tropomyosin-binding sites at both the N- and C-terminal domains of caldesmon, we have studied the binding of the N- and C-terminal fragments of human fibroblast caldesmon expressed in Escherichia coli to tropomyosin and its CNBr fragments. The N-terminal fragment, CaD40 (residues 1-152), binds tropomyosin, but the interaction is mostly abolished in the presence of actin. CaD40 binds strongly to Cn1B(142-281) of tropomyosin, but weakly to Cn1A(11-127). The C-terminal fragment, CaD39, which corresponds to residues 443-736 of gizzard caldesmon, binds tropomyosin, and the interaction is enhanced by actin. CaD39 binds to both Cn1A(11-127) and Cn1B(142-281) of tropomyosin. Our results suggest that the N-terminal domain of caldesmon interacts with the C-terminal half of one tropomyosin molecule, whereas the C-terminal domain binds to both N- and C-terminal regions of the adjacent tropomyosin molecule along the actin filament. In addition, the binding of the N-terminal domain of caldesmon to the actin-tropomyosin filament is weak, which may allow this domain to project off the thin filament to interact with myosin.

Actins↗

PubMed1995

Use of preputial skin for the release of burn contractures in children.

Contractures are frequent sequelae of burn injuries. Progressive worsening of contractures with limitation of joint movement is common in children. The results of release procedures in 10 male Chinese children using their own preputial skin obtained from circumcision were reviewed. This technique yields reliable results and is well accepted by the parents.

Adolescent↗

PubMed1995

Regional myosin heavy chain expression in volume and pressure overload induced cardiac hypertrophy.

Although the overall shift towards the V3 myosin heavy chain (MHC) has been shown to be associated with cardiac hypertrophy, quantitative evidence describing regional expression is sparse. The aim of this study was to compare and contrast the regional ventricular myosin isoform expression in two distinct haemodynamic states: pressure and volume overload. Volume overload was achieved using an aortocaval fistula (ACF) model and pressure overload by two-kidney-one-clip (2K1C) hypertension. A separate group (UC-2K1C) had the clip removed 1 week prior to investigation. Sham operated rats (SHAM) served as controls. All groups were studied 4 weeks after surgery. Ventricular tissue samples (approximately 50 mg) were taken from the walls of the right ventricle (RV), septum and left ventricular (LV) free wall. Tissue samples (excluding RV) were divided into endocardium and epicardium, and myosin expression was determined using polyacrylamide gel electrophoresis. Cardiac hypertrophy was substantial in both LV (1.7-fold) and RV (1.9-fold) in ACF rats. The 2K1C rats had similar LV enlargement (1.6-fold) whereas RV hypertrophy was not as great (1.2-fold). Blood pressure (BP) was increased 65% in 2K1C rats, whereas there was no change in ACF rats with respect to SHAM animals. After unclipping (UC-2K1C), LV hypertrophy and BP had returned towards control levels. In general, V3 MHC expression was associated with increasing LV hypertrophy in both 2K1C and ACF models. However, there was a marked endo-epi differential (1.5:1) in the LV free wall and septum of 2K1C rats. In contrast, in ACF rats there was no differential V3 MHC expression in the LV or septal tissue, i.e. expression was similar in both endo- and epi-samples. Elevated expression of V3 MHC persisted despite normotension and regression of cardiac hypertrophy in UC-2K1C rats. Taken together with published results demonstrating that relative transmural myocyte hypertrophy in ACF rats (endo > epi) is in contrast to that seen in 2K1C rats (epi > endo), the present findings reveal that regional V3 myosin expression represents a distinct adaptational component of the overall cardiac hypertrophic response in both volume and pressure overload.

Animals↗

PubMed1995

Audit of basal cell carcinoma in Princess Margaret Hospital, Hong Kong: usefulness of frozen section examination in surgical treatment.

A retrospective study was undertaken of 64 Chinese patients with primary (previously untreated) basal cell carcinoma (BCC) surgically treated by the plastic and reconstructive surgery team at the Princess Margaret Hospital, Hong Kong, from January 1988 to March 1994. Sixty-three (98%) were in the head and neck region, half on the nose. It was equally common in men (mean age 69 years) and women (mean age 67). Two women (3%) were younger than 35. The rate of complete excision increased after the introduction of frozen section examination. A complete excision rate of 89% (n = 57) was achieved by 1994. We conclude that frozen section examination should be done routinely in patients having BCC excised.

Adult↗

PubMed1993

Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.

A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity.

Amino Acid Sequence↗

PubMed1993

Differential expression and activity of p34cdc2 in cultured aortic adventitial fibroblasts derived from spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: The present investigation was undertaken to determine whether p34cdc2, a cell-cycle regulatory kinase, is involved in the manifestation of the altered proliferation evident in fibroblasts isolated from spontaneously hypertensive rats (SHR). DESIGN: Experiments were performed on quiescent aortic adventitial fibroblasts stimulated to re-enter the cell cycle in order to examine the timing of cell cycle-related events. METHODS: The cell-cycle phase was determined by flow cytometry and was related to the cellular content and kinase activity of p34cdc2. RESULTS: SHR fibroblasts displayed a heightened basal level of p34cdc2 at quiescence relative to Wistar-Kyoto (WKY) rat cells. Both SHR and WKY fibroblasts showed a cell cycle-dependent increase in p34cdc2 content, beginning in S phase. However, the SHR adventitial fibroblasts exited G0-G1 several hours earlier than the WKY fibroblasts as indicated by the time of initiation of DNA synthesis and increase in activity of p34cdc2. CONCLUSIONS: SHR aortic adventitial fibroblasts appear to have a heightened proliferative capacity relative to WKY fibroblasts, which is evident in a quicker exit from G0 and faster transition to DNA synthesis, followed by the earlier activation of p34cdc2.

Animals↗

PubMed1993

Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.

Animals↗

PubMed1992

Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells.

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.

Amino Acid Sequence↗

PubMed1992

Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.

Animals↗