The stimulation of calcium uptake into sarcoplasmic-reticulum vesicles from rat heart by adenosine 3',5'-phosphate-dependent protein kinase [proceedings].

There is now considerable evidence that the increase in contractility that occurs in heart muscle in response to catecholamines is mediated through changes in cyclic AMP (Sobel & Mayer, 1973). It has been proposed that the increase in cyclic AMP stimulates phosphorylation of a protein associated with the sarcoplasmic reticulum, causing an increase in Ca2+ uptake and a subsequent redistribution of Ca2+ in the heart (Katz et al., 1975). Incubation of isolated sarcoplasmic-reticulum vesicles with cyclic AMP-dependent protein kinase caused an increase in Ca’+ uptake (oxalate-dependent and -independent), providing evidence for this hypothesis (Tada et al., 1974). This was associated with phosphorylation of a protein of mol.wt. 23000. It was reported (Gibson & Newcomb, 1975) that sarcoplasmic-reticulum vesicles from rat heart were not phosphorylated by added protein kinase, unlike those from the hearts of dog, rabbit and guinea pig. In the present communication we show that with purified vesicles from rat heart, added protein kinase caused an increase in oxalate-dependent Ca2+ uptake, and that this was associated with phosphorylation of two proteins of mol.wts. 23000 and 17000. Sarcoplasmic-reticulum vesicles were prepared essentially as described by Levitskii et al. (1976). Fresh rat hearts (approx. 3g) were homogenized (Polytron PTlO homogenizer) in 15 ml of 0.29~-sucrose/5 m~-NaN,/30m~-Tris/HCl/l5 mhl-2-rnercaptoethanol/O.Srn~-EDTA, pH8.0, at 4”C, and centrifuged at IOOOOg for 20min. The supernatant was centrifuged at 28000g for 90min and the resultant pellet resuspended in 2ml of I.l7~-sucrose, IOmM-Tris/HCI, pH7.2, at 4°C (buffer A) and diluted with 2ml of 1 . ~ M K C ~ / ~ ~ M E G T A / ~ ~ M A T P / ~ ~ ~ M M ~ C ~ ~ / ~ O ~ M T ~ ~ ~ / H C I , pH 7.8, at 4°C. This was incubated at 4°C for 40min to solubilize myofibrillar proteins, and centrifuged at 8OOOOg for 90min. The pellet was resuspended in 2ml of buffer A. With dog and other species this method produces a relatively clean sarcoplasmic-reticulum preparation. However, with rat heart there was contamination with Ca2+-insensitive ATPase* (see also Levitskii el a[., 1976) that prevented studies on protein phosphorylation. Further purification was therefore carried out by partially loading the vesicles with calcium oxalate followed by centrifugation in a sucrose density gradient. The preparation was diluted with 2ml of 120m~-ATP/10m~-~a~~/~OOm~-KC1/10m~-potassium oxa~ate/20m~-Mg~~~/0.1~m~-CaC~,/~Orn~-Tris/~c~, pH7.2, at 2 0 T , and incubated for 30min at 37°C. This was layered on to a discontinuous sucrose/KCI density gradient (see Fig. 1 ) and centrifuged at 8OOOOg for 90min. Caz+ uptake into sarcoplasmic-reticulum vesicles in the presence of oxalate was measured by filtration on Millipore membranes as described by Harigaya & Schwartz (1969), with 4sCaC12 in the absence of EGTA. Fig. l (a) shows the distribution of 45Ca in the fractions obtained after sucrose-densitygradient centrifugation when the loading procedure was carried out with 4sCaCI,. It can be seen that most of the bound 4sCa, as assayed by membrane filtration, was in the pellet. Fig 1 (b) shows the distribution of protein after sucrose-density-gradient centrifugation, with and without preloading with calcium oxalate. Without preloading there was no pellet, and assay of the fractions for ability to accumulate CaZ+ showed that nost of this activity was present at the (i)/(ii) and (i i ) / ( i i i ) interfaces (see Fig. Ib). After preloading, a pellet was found that retained considerable ability to accumulate CaZ+, and there was a decrease in this activity at the (i)/(ii) and (ii)/(iii) interfaces. The pellet had an ATPase activity of 0.1 pmol/min per mg of protein at 30°C, which was approximately * Abbreviation: ATPase, adenosine triphosphatase.