A model for the coregulation of smooth muscle actomyosin by caldesmon, calponin, tropomyosin, and the myosin regulatory light chain

1994 ◽  
Vol 72 (11) ◽  
pp. 1400-1409 ◽  
Author(s):  
Joe R. Haeberle ◽  
Mark E. Hemric
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Regulatory Protein ◽  
Regulatory Light Chain ◽  
Motility Assay ◽  
Actin Binding ◽  
Shortening Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

The purpose of these studies was to evaluate the effects of the actin-binding proteins tropomyosin, caldesmon, and calponin on the activation of smooth muscle actomyosin by phosphorylation of the regulatory light chain of myosin (LC20), and to interpret these findings in the context of a two-state kinetic model for the cross-bridge cycle. An in vitro motility assay was used to broadly classify each regulatory protein according to whether it modulates the apparent on-rate for cross bridges (fapp) or the apparent off-rate (gapp). In addition to measuring actin-filament velocity, a method was developed to measure relative changes in the force exerted on actin filaments under isometric conditions. Based primarily on the results of these motility studies, a qualitative model is proposed in which LC20 phosphorylation, tropomyosin, and caldesmon all regulate fapp and calponin regulates gapp. The model predicts that the sensitivity of activation by LC20 phosphorylation is determined by tropomyosin, caldesmon, and calponin, whereas unloaded shortening velocity is regulated primarily by calponin.Key words: smooth muscle, caldesmon, calponin, tropomyosin, motility assay.

The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay

2012 ◽  
Vol 113 (9) ◽  
pp. 1413-1422 ◽  
Author(s):  
Edward P. Debold ◽  
Thomas J. Longyear ◽  
Matthew A. Turner
Keyword(s):  
Molecular Basis ◽  
Bound States ◽  
Contractile Activity ◽  
Motility Assay ◽  
Shortening Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Specific Step ◽  
Regulated Thin Filament

Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (Pi) and hydrogen ions (H+) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H+ and Pi on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca++, acidosis depressed regulated filament velocity ( VRTF) by ∼90% (6.2 ± 0.3 vs. 0.5 ± 0.2 μm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM Pi caused VRTF to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 μm/s at pH 6.5. Similarly, at all subsaturating Ca++ levels, acidosis slowed VRTF, but the addition of Pi significantly attenuated this effect. We also manipulated the [ADP] in addition to the [Pi] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H+. The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to Pi rebinding, and the associated detachment from actin, this finding may also explain the Pi-induced enhancement of VRTF at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.

Phosphorylation of calponin in airway smooth muscle

1997 ◽  
Vol 272 (1) ◽  
pp. L115-L123 ◽  
Author(s):  
J. Pohl ◽  
S. J. Winder ◽  
B. G. Allen ◽  
M. P. Walsh ◽  
J. R. Sellers ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Inhibitory Effect ◽  
Tracheal Smooth Muscle ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Kinase C ◽  
Gradient Gel Electrophoresis

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.

Characterization of isoform diversity in smooth muscle myosin heavy chains

1994 ◽  
Vol 72 (11) ◽  
pp. 1351-1360 ◽  
Author(s):  
Christine A. Kelley ◽  
Robert S. Adelstein
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Smooth Muscle Myosin ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Amino Terminal ◽  
Muscle Myosin

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3′ end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC204 and the shorter, 200-kDa isoform as MHC200. We found that MHC204, but not MHC200, can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC204 and MHC200 form homodimers, but not heterodimers. Purified MHC204 and MHC200 homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC204 and MHC200 isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC204-I; MHC200-I) or noninserted (MHC204; MHC200), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.Key words: myosin, heavy chain isoforms.

Caged FEDA-ATP: a new tool in the measurement of ATP turnover during the in vitro motility assay

1995 ◽  
Vol 23 (3) ◽  
pp. 401S-401S ◽  
Author(s):  
Daren S. Jeffreys ◽  
Robert J. Eaton ◽  
Clive R. Bagshaw
Keyword(s):  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Atp Turnover
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