Cloning and expression of an insect Ca2+-ATPase from Heliothis virescens

AbstractA complementary DNA for the Tobacco Budworm, Heliothis virescens, sarco(endo)plasmic reticulum-type Ca2+-ATPase (HVSERCA) has been cloned and sequenced. cDNA fragments of adult rabbit fast-twitch muscle Ca2+-ATPase (SERCA1a) were used as heterologous probes to isolate a partial cDNA clone coding for a protein with high homology to the Ca2+-ATPase from Drosophila melanogaster (DRSERCA) and vertebrate ER/SR Ca2+ pumps. The entire cDNA clone contains an ORF encoding a protein of 1000 amino acids which shares the characteristic motifs of a P-type ATPase. HVSERCA shares 89% identity with DRSERCA, 80% identity with the Artemia Ca2+-ATPase and 72% identity with avian and mammalian SERCAs. An insect Ca2+-ATPase-specific polyclonal antiserum has been raised against a fusion protein containing sequence from the cytoplasmic domain of HVSERCA. Heterologous expression of the insect pump in COS-7 cells has been demonstrated by immunocytochemistry and the reticular pattern of staining is consistent with an ER localisation. However, the expressed enzyme from COS-7 cells does not appear to be active

The presence of sarcolipin results in increased heat production by Ca2+-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane -helix. When reconstituted with the Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca2+. Heats of reaction of the reconstituted Ca2+-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca2+-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN·ATPase complex of 6.9 x 10–4 ± 2.9 x 10–4 in units of mol fraction per monolayer. It is suggested that the interaction between Ca2+-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum