INFRARED RAMAN AND COMPOSITION STUDIES OF SKELETAL MUSCLE SARCOPLASMIC RETICULUM (MEMBRANE, PROTEIN, LIPID, CALCIUM-ATPASE, TWITCH)

Since the kinetics of the Ca('2+)-ATPase varies with the membrane composition of the sarcoplasmic reticulum (SR), it was of interest to make a comparative study of the SR membrane components from fast and slow twitch muscle. Because of small sample size and the inaccuracy of current chemical methods, we developed a quicker, and more accurate method of determining lipid/protein ratios using the IR protein amide stretch and the lipid carbonyl band. With this technique, we demonstrated that fast twitch muscle contains more Ca('2+)-ATPase/lipid in SR than slow twitch muscle.;All aspects of the soleus SR composition compared to caudofemoralis SR composition including high protein, cholesterol, phosphatidylethanolamine, sphingomyelin content and lipid saturation favor a more ordered lipid membrane, and hence decreased Ca('2+)-ATPase activity for soleus SR.;Raman and infrared temperature studies of fast and slow twitch muscle Ca('2+)-ATPase purified SR vesicles or SR lipid dispersions showed that above -10(DEGREES)C the soleus SR membrane is more ordered than caudofemoralis SR membrane as the composition studies suggest.;The Ca('2+)-ATPase protein disordered the endogenous caudofemoralis SR lipid and shifted the order-disorder transitions centered at 0.3(DEGREES)C and 17(DEGREES)C to -3 and 13(DEGREES)C respectively. The transition centered at -9(DEGREES)C remained unchanged.;IR and Raman spectroscopy was also used to show that the soleus (slow twitch) SR is twice as saturated as caudofemoralis (fast twitch) SR lipid.;Concomitant with these lipid transitions were a number of protein structural changes detected by Raman spectroscopy. The average typtophan environment becomes more hydrophobic near 17(DEGREES)C for combined muscle SR, and a decrease in the number of caudofemoralis SR Ca('2+)-ATPase tyrosine residues being strongly hydrogen bonded to the interior of the Ca('2+)-ATPase protein is observed between 10 and 14(DEGREES)C. These structural changes correspond to the break in the arrhenius plot of Ca('2+)-ATPase activity measured near 14(DEGREES)C and the center of the lipid order-disorder transition. The soleus SR showed no breaks in the arrhenius plot of Ca('2+)-ATPase activity or sharp lipid order disorder transitions.;A decrease of intensity in the 3065 cm('-1) aromatic amino acid band at 2(DEGREES)C for caudofemoralis SR is interpreted by model studies to be due to an increase in the hydrophylic environment of the protein.