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The major form of calsequestrin in rabbit slow‐twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast‐twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast‐twitch calsequestrin cDNA probe to mRNAs isolated from fast‐twitch and slow‐twitch muscles; identity of the 23 amino‐terminal amino acids; strong binding of 45Ca2+in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast‐twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast‐twitch muscle.

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b5were coexpressed with the Ca2+‐ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four‐base circuit.

The nucleotide sequences of cDNAs encoding phospholamban were found to be virtually identical when the cDNA clones were isolated from rabbit slow-twitch (soleus) and rabbit cardiac muscle libraries. These findings demonstrate that both types of muscle express the same phospholamban gene. The deduced amino acid sequences of rabbit and dog phospholamban were identical except for a change from Asp (dog) to Glu (rabbit) at position 2. The nucleotide sequences of the 5′- and the very long 3′-untranslated regions of rabbit and dog phospholamban cDNAs also exhibited a high percentage of identity.

A calmodulin-dependent protein kinase system from the sarcoplasmic reticulum was dissolved in Nonidet P40, adsorbed to a CaM affinity column in the presence of Ca 2+and eluted in the presence of EGTA. The purified fraction contained major proteins of 60 and 20 kDa and minor components of 89 and 34 kDa, all of which were phosphorylated with dependencies on Ca 2+, CaM, ATP and pH similar to those observed in the sarcoplasmic reticulum. Differences in the phosphopeptides produced by partial proteolysis of the individual phosphoproteins indicated that they are distinct entities. 125I-CaM labeled only the 60 kDa protein, suggesting that it is a kinase.

We have assessed the effects of glutamine (Gln) availability on protein breakdown in perfused rat hindlimb by measuring net phenylalanine (Phe) production (an index of protein balance), the dilution of [ 15N]Phe labelling (an index of mixed protein breakdown) and rate of production of 3-methylhistidine (3-MeH) (an index of myofibrillar breakdown). 15 mM Gln significantly inhibited net protein loss and protein breakdown compared to rates obtained in its absence (net protein loss, 200±230 vs 2080±200 nmol Phe/hindlimb per h; protein breakdown, 4566±480 vs 1614±180 nmol Phe/hindlimb per h; both p<0.01). Insulin (100 μU/ml) inhibited protein breakdown but less than Gln. The effects on protein breakdown of Gln and insulin together were not additive, suggesting a common mode of action. Production of 3-MeH (mean 20.3±2.8 nmol/hindlimb per h) was unaffected by Gln or insulin. Gln appears to inhibit protein breakdown of soluble rather than myofibrillar protein in muscle.

During muscle-protein wasting associated with injury and disease the distribution ratio of free glutamine between muscle and blood falls. In pursuing possible consequences of this, we investigated the relationship between the rate of muscle protein synthesis and intramuscular glutamine concentration, manipulated acutely in the isolated perfused rat hindquarter. Increasing perfusate glutamine from 0.67 to 5.0 mM caused a 200% increase in intracellular glutamine and a 66% increase in protein synthesis in the absence of insulin; in the presence of insulin a 30% increase in intramuscular glutamine was accompanied by an 80% increase in protein synthesis. Analysis of variance of the results confirmed the existence of positive relationships between intramuscular glutamine and protein synthesis in the presence or absence of insulin. Control of the size of the intramuscular free pool of glutamine may be important in determining the muscle protein mass.

Full length cDNAs encoding both slow-twitch/cardiac (SERCA2) and fast-twitch skeletal muscle (SERCA1) Ca2+-ATPases were expressed by transient transfection of COS-1 cells. Studies of the Ca2+-dependency of Ca2+-transport in microsomes isolated from these cells showed that both isoforms had an affinity for Ca2+of about 0.2 μM. The Ca2+-affinity of SERCA2 was lowered when phospholamban was co-expressed with it, demonstrating that the two proteins interact in this expression system. These studies support the view that phospholamban inhibition accounts for the low Ca2+-amnity and low activity of SERCA2 in cardiac muscle sarcoplasmic reticulum.

Deletion mutants of the Ca 2+ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M 1and M 2, but not of M 8, M 9, M 10or the NH 2 −terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M 1and M 2.

Deletion mutants of the Ca2+ATPase of rabbit fast‐twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS‐1 cells. The mutants were expressed at levels 7‐ to 15‐fold lower than the wild‐type and were inactive. In vitro transcription‐translation‐insertion experiments showed that deletion of transmembrane sequences M1and M2, but not of M8, M9, M10or the NH2−terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1and M2.