Open Collections

Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum

University of British Columbia

Calmodulin prepared from red cell hemolysate stimulates Ca²⁺ -transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum (S.R.) in a concentration-dependent manner (Katz and. Remtulla, 1978). The present study was performed to characterize this calmodulin regulation of Ca²⁺-transport. K⁺ and Na⁺ have also been found to enhance Ca²⁺-transport in microsomal preparations enriched in S.R. (Jones et al, 1978). It was observed that in the presence of K⁺ (110 mM) and Na⁺ (110 mM) stimulation of Ca²⁺-transport activity by calmodulin was greatly reduced. This result was obtained at all free Ca concentrations studied. That this was not an ionic effect was indicated by the decreased antagonism of calmodulin stimulation by Li⁺ (110 mM). Kinetic characterization determined that calmodulin significantly enhanced the maximal Ca²⁺activation without affecting the apparent Ca²⁺affinity of the Ca²⁺-transport process in cardiac S.R.. K⁺ was found to enhance the V[sub Ca²⁺] as well as lowering the apparent Ca²⁺ affinity. Examination of the initial rate of Ca²⁺-transport in cardiac S.R. confirmed that calmodulin stimulation is due mainly to an increase in the V[sub Ca²⁺]. CyclicAMP-dependent protein kinase, on the other hand, has been shown to increase the V[sub Ca²⁺], as well as decrease the apparent K[sub m] for Ca²⁺ (Hicks et al, 1979), suggesting a different mechanism of action. Experiments were performed to investigate whether calmodulin was indigenous to the preparations used. Attempts were therefore made to isolate calmodulin from dog cardiac microsomal preparations enriched in S.R. by methodology used to isolate calmodulin from other sources (Jung, 1978; Depaoli-Roach et al, 1979). These extracts were then tested to determine their ability to stimulate Ca²⁺-transport into S.R.. It was observed that neither boiling nor treatment with 0.6mM EGTA could extract calmodulin from these preparations. This result indicates that the microsomal preparations utilized do not contain indigenous calmodulin. Since calmodulin dose not appear to be a component of the S.R., it was postulated that binding to sites on the membrane must occur in order for calmodulin to augment Ca²⁺- transport. It was also suggested that K⁺ and NA⁺ and may decrease calmodulin stimulation of Ca²⁺-transport by altering this binding. Studies were therefore performed using ¹²⁵I-labelled calmodulin to determine the degree of binding to microsomal preparations in the presence and absence of K⁺(110mM), Na⁺ (110mM) , and Li⁺(110mM.). It was found that ¹²⁵I-calmodulin binds to microsomal cardiac S.R. in a Ca²⁺ concentration-dependent manner. At Ca²⁺ concentrations above 10⁻⁷M, this binding was significantly decreased (p<0.05, students "t" test) in the presence of K⁺ or Na⁺. Li⁺, previously shown not to alter Ca²⁺-transport augmentation byucalmodulin, did not alter calmodulin binding to a significant extent. K⁺ and Na⁺ therefore may inhibit calmodulin stimulation of Ca²⁺-transport in these preparations by decreasing calmodulin binding to the S.R.. The lack of inhibition by Li⁺ (110mM) indicates that this result is not due to a non-specific ionic effect. Our studies therefore have shown that calmodulin is not an indigenous protein of the sarcoplasmic reticulum preparations used. Calmodulin, though, has been found to bind, in a Ca²⁺-dependent manner, to these preparations. This binding is altered by monovalent cations previously shown to inhibit calmodulin stimulation of Ca²⁺-transport.

Text

2010-03-19

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/22153

Pharmaceutical Sciences

University of British Columbia

Graduate