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Biomedical subjects

J Gulati

Publications and source records attributed to J Gulati.

At least 37 records · Page 2Linked to original sources
PubMed1992

Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.

Utilizing protein engineering of troponin C (TnC), combined with the physiology of skinned fibers, the present study sought to delineate the mechanisms for metal ion coordination and sensitivity in the sites (EF-hands) that execute the Ca2+ switch for contraction. A total TnC-encoding gene comprising multiple target sequences for restriction enzymes was synthesized, furnishing a pliant molecular handle to manipulate sites I and II in the NH2 terminus of the protein. Of the six positions (X, Y, Z, -Y, -X, and -Z) essential for metal ion chelation in a typical EF-hand, invariably the X position has aspartate, and -Z position has glutamate. In the X position of site II, mutation of aspartate for either glutamate (gamma-carboxylate) or asparagine (same side chain length as aspartate) yielded functionally inactive proteins with concomitantly diminished Ca2+ binding capacity. Similarly, in -Z position (site I), neither aspartate nor glutamine were compatible in exchange for the conserved glutamate. In contrast, for the Y coordinate of site II, a preference for asparagine comparable to that for wild-type aspartate was detected, but glutamate was impermissible. Evidently, physicochemical and steric factors both are critical in governing the mechanism for metal ion chelation in TnC in a physiological milieu. Furthermore, the findings manifest that the quaternary structure of hydrated TnC restrains the EF-hands during on-off operation of the Ca2+ switch.

Amino Acid Sequence↗
PubMed1991

The role of troponin C in the length dependence of Ca(2+)-sensitive force of mammalian skeletal and cardiac muscles.

1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2+ and the length dependencies of their pCa (-log [Ca2+])-force relationships were compared. 2. Ca2+ sensitivity of the myocardium was higher at 2.2-2.4 microns than that at 1.7-1.9 microns. The length dependence was at least twofold greater in cardiac muscle than in fast skeletal fibres at identical temperatures and salt concentrations. Slow-twitch fibres gave a response similar to that in the myocardium. 3. The effect of the troponin C (TnC) phenotype on the length dependence of Ca2+ sensitivity was measured on both fast skeletal fibres and cardiac muscle with TnC exchange in situ. The length-induced increase in Ca2+ sensitivity was found to be greater in the presence of cardiac TnC than with fast skeletal TnC. Thus the results indicate that a certain domain of TnC is specialized in this length function, and that this domain is different in the two phenotypes. 4. The possibility that the enhanced length dependence of Ca2+ sensitivity after cardiac TnC reconstitution was attributable to reduced TnC binding was excluded when the length dependence of partially extracted fast fibres was reduced to one-half the normal value after a 50% deletion of the native TnC. 5. Two recombinant forms of cardiac TnC (kindly provided by Dr John Putkey, Houston, TX, USA) were used next, to investigate the roles of two specific domains in TnC in the control of length dependence of Ca2+ sensitivity and in the contraction-relaxation switching of cardiac muscle: 6. Using mutant CBM1 [corrected], in which site 1 was modified such as to bind the 4th Ca2+ ion, as in skeletal TnC, the length-induced Ca2+ sensitivity in cardiac muscle was suppressed. The effect was intermediate between cardiac and skeletal TnCs under the same conditions. The pSr (-log [Sr2+])-force relationship of cardiac muscle was also measured. In the presence of the mutant, skinned trabeculae manifest pSr-activation curves identical to those of fast fibres. This indicates that the metal ion binding properties of site 1 in TnC modulate the regulatory action of site 2. 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin.

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.

Animals↗
PubMed1990

Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles.

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.

Animals↗
PubMed1989

Characterization of the Ca2+-switch in skeletal and cardiac muscles.

To determine the significance of the global structure of the regulatory proteins in the mechanism of the Ca2+-switch in cardiac and skeletal muscle contractions, the properties of a family of Ca2+-binding proteins with 4 or 3 EF-hand motifs have been studied with desensitized skinned fiber preparations. Proteins with 4 EF hands (such as troponins C - TnCs) are dumb-bell shaped, those with 3 EF hands (parvalbumin) being ellipsoidal. The number of active sites varied between four and two. We find that the ability to anchor in the fiber is limited to proteins with 4 EF hands and, at least, two active Ca2+-binding sites, one each in the N- and C-termini. The results suggest that the dumb-bell shaped global structure is critical for the switching action in muscular contraction, and a trigger site in the N-terminus and a structural site in the C-terminus need to be active in order to regulate contractility.

Animals↗
PubMed1989

Down-regulation of fast-twitch skeletal muscle fiber with cardiac troponin-C and recombinant mutants. Structure/function studies with site-directed mutagenesis.

Structure/function relationships in troponin C are studied with vertebrate fast-twitch fibers by exchanging the skeletal troponin C with two bacterially synthesized recombinant proteins designed by site-directed mutagenesis of cardiac troponin C. One mutant (CBM1) contained an additional active site, by deleting Val-28 and converting Leu-29, Gly-30, Ala-31 and Glu-32 to Asp, Ala, Asp and Gly, respectively, in the normally inactive trigger site 1 in the N-terminus. In another mutant (CBM2A), the normally active site 2 was inactivated by conversion of Asp-65 to Ala. The fibers were found to be down-regulated with recombinant cardiac troponin C (CTnC3), as with tissue-cardiac-troponin-C. With mutants, in one case (CBM1) the regulation was unmodified despite Ca2+ coordination by all sites. In contrast, regulation was found to be completely blocked with the mutant (CBM2A) where both trigger sites were inactive. The results provide the first indication that structural specification of the entire EF-hand motif of site 1, and not just Ca2+ coordination, is needed to operate fully the Ca2+ switch in fast-twitch fibers.

Animals↗
PubMed1989

Effect of acidosis on Ca2+ sensitivity of skinned cardiac muscle with troponin C exchange. Implications for myocardial ischemia.

By using a novel approach for the study of the effects of pH variation in skinned myocardium, the present experiments were aimed to provide new insights into the mechanism of ischemia. Ca2+ sensitivity is decreased by acid pH, but the effect is more than double in cardiac myofilaments than that in fast-twitch skeletal muscle fibers. With the technique of troponin C exchange in myocytes, we find here that the effect of pH is the same with cardiac or skeletal troponin C. These results rule out a direct H+-Ca2+ competition on the Ca2+-binding sites of troponin C as a significant mechanism of ischemia. The findings provide conclusive evidence in favor of the idea that acidosis modulates the protein-protein interactions in the regulatory complex in cardiac muscle.

Animals↗
PubMed1988

Calmodulin supports the force-generating function in desensitized muscle fibers.

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.

Animals↗
PubMed1988

Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle.

This study describes the effects of exchanging native cardiac troponin C (CTnC) from the right ventricular muscle of Syrian hamster for purified skeletal (S) TnC from fast twitch muscles in triggering cardiac contraction. Ca2+ sensitivity of the myocardium became decreased with STnC to 62% of the original value with CTnC. Furthermore, the slope of the pCa-force curve of cardiac muscle was found to be increased with STnC. The results show that the TnC moiety, as part of the switching mechanism during activation, also regulates thin-filament cooperativity in muscle. Modifications in both the Ca2+ sensitivity and cooperativity are associated with alterations in the primary structure of TnC.

Animals↗
PubMed1988

Molecular basis for the influence of muscle length on myocardial performance.

According to Starling's law of the heart, the force of contraction during the ejection of blood is a function of the end-diastolic volume. To seek the molecular explanation of this effect, a study was made of the effects of length on Ca2+ sensitivity during tension development by isolated demembranated cardiac muscle in which the cardiac form of troponin C was substituted with skeletal troponin C. The results of troponin C exchange were compared at sarcomere lengths of 1.9 and 2.4 micrometers. Enhancement of the myocardial performance at the stretched length was greatly suppressed with the skeletal troponin C compared with the cardiac troponin C. Thus the troponin C subunit of the troponin complex that regulates the activation of actin filaments has intrinsic molecular properties that influence the length-induced autoregulation of myocardial performance and may be a basis for Starling's law of the heart.

Animals↗
PubMed1988

Evidence for novel 30,000-50,000Mr cofactor in the activation of muscle.

A new approach is described for reconstituting a fully desensitized skeletal muscle fiber to restore its contractility. These studies revealed a novel regulatory cofactor, 30-50,000Mr by filtration (26-55kDa by SDS PAGE). It was shown to be critical for the Ca2+-activation in the physiological milieu. The cofactor was present in skeletal and cardiac muscles as well as in brain, but not in kidney and liver. The cofactor may be a second Ca2+ switch in a dual-regulation scheme for vertebrate muscle, or could provide an essential link in the cross-bridge cycle beyond activation.

Animals↗
PubMed1988

Proposed mechanism for dual regulation of cross-bridge turn-over in vertebrate muscle.

The studies on intact fibers of the frog and fast-twitch skinned fibers of the Syrian hamster suggest the presence of intermediate low-force (weak) and high-force (strong) attachments in the cross-bridge cycle. Our results suggest that the weak and strong attachments may be separately regulated by independent calcium switches. The switch for weak bridges operates by calcium or decreasing ionic strength, such that in low ionic strength in skinned fibers the bridges form even in the virtual absence of TnC and calcium. The weak to strong transition appears to require Ca in both low and high ionic strengths. The Ca action effecting this transition may be mediated also by TnC and/or may involve the myosin moiety. Thus, the possibility is raised that Ca may act at two separate steps in the cross-bridge mechanism under physiological conditions.

Actins↗
PubMed1987

The control of myocardial contraction with skeletal fast muscle troponin C.

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.

Animals↗
PubMed1986

Ca2+ activation of troponin C-extracted vertebrate striated fast-twitch muscle fibers.

To characterize the tension control in vertebrate striated muscle fibers, and to obtain insights into the cross-bridge mechanisms, Ca2+ activation on troponin C (TnC)-extracted skinned fibers was studied in standard (180 mM, physiological) and low (20-41 mM) ionic strength solutions. By tension measurement, TnC-extracted fibers had nearly lost their Ca2+ sensitivity in the standard ionic strength solutions, but surprisingly the fiber still exhibited significant tension on activation with Ca2+ in low ionic strength. Also, the presence of weak bridges (zero-force bridges) was inferred by stiffness measurements in Ca2+-free low ionic strength solution, and were found even after TnC extraction. The possibility is discussed that dual regulation by Ca2+ is present in the vertebrate muscle. One mechanism activates the thin filaments. The second may directly control the kinetic step for the transition between the weak and strong bridges, in the cross-bridge cycle in the fiber, and in this way may act as an additional Ca2+ switch.

Animals↗
PubMed1986

Kinetics of force redevelopment in isolated intact frog fibers in solutions of varied osmolarity.

Isolated intact frog muscle fibers, while shortening with the intrinsic maximal speed, were stretched back to the original length to measure the kinetics of force redevelopment. These kinetics give information on the attachment rate constant in the cross-bridge cycle in vivo, and a value of approximately 25.6 s-1 (0 degree C) is found in the present study. We find that these kinetics were slightly less sensitive to temperature than was the unloaded shortening speed. The effect of hyperosmolarity on force redevelopment was also measured in solutions with added sucrose or KCl. The rate constant was nearly halved with 120 mM sucrose, but there was practically no effect with isosmotic (60 mM) KCl. These results indicate that the rate constant of force redevelopment is insensitive to raised intracellular ionic strength. In sucrose, the fiber width was also compressed, and the attenuation of the rate constant of force redevelopment in this case is consequently attributed to the decrease in interfilament space. The order of magnitude of the rate constant found in this study suggests that tension transduction by a cross-bridge, during each turnover cycle, requires a series of elementary steps following the attachment.

Animals↗
PubMed1985

Critical dependence of calcium-activated force on width in highly compressed skinned fibers of the frog.

Force development by skinned frog semitendinosus fibers was studied at various levels of lateral compression to compare the results with intact fibers and to evaluate the limits on cross-bridge movements during isometric contraction. The skinned fibers were compressed osmotically using a high molecular weight polymer, dextran T500. Ca-activated force remained constant down to 58% of the fiber width (w0) after skinning, corresponding to a nearly twofold change in separation between the thin and thick filaments in the myofilament lattice. This agrees with the earlier result on intact fibers, and gives additional evidence that the cross-bridge mechanism for force generation is relatively insensitive to large changes in interfilament separation. Further compression, below 0.58 w0, produced a sharp drop in force, and the force was practically zero at a fiber width of 50%. The effect at high compression was the same at all pCa's, which indicates that the Ca sensitivity of the myofilaments is unaffected by radial compression. The stiffness of the fiber remained high in rigor in the presence of dextran, which indicates that the rigor cross-bridge attachment is not inhibited, and actually may be improved, with decreases in the interfilament space. Also, the drop in active force with the highest compression was similar when the compressed fibers were put in rigor before contraction, which suggests that the force drop also was not due to a hindrance to cross-bridge attachment. The results appear to exclude large motions such as tilting and rocking of the bridge as a rigid molecule, but suggest that at least some molecular movement is essential for force development; they also raise the possibility that there is a critical interfilament separation in the fiber, below which the cross-bridge cannot function.

Animals↗