Taibo Yamamoto

Reaction Mechanisms for ATP Hydrolysis and Synthesis in the Sarcoplasmic Reticulum

Publisher Summary This chapter focuses on the reaction mechanisms for ATP hydrolysis and synthesis in the sarcoplasmic reticulum. The membrane system in muscle cells, which has a role in controlling muscle contraction, consists of the plasma membrane with its tubular infoldings running transversely to the fiber axis and a reticular structure that forms a network surrounding the myofibrils. The fragmented sarcoplasmic reticulum (FSR) offers many advantages for studying the molecular mechanism of active transport. FSR can easily be isolated in large quantities from muscle homogenates. ATPase protein accounts for more than two-thirds of the total protein in the FSR membrane. The ATPase reaction is tightly coupled to Ca 2+ transport; 2 mol of Ca 2+ are transported for each mole of ATP hydrolyzed under a variety of conditions. The sidedness of the FSR membrane where the enzyme reacts with the substrate, products, and cation can be clearly distinguished. The concentrations of Ca 2+ and Mg 2+ outside the membrane can be controlled with chelators, and the concentration of Ca 2+ inside the vesicle can be controlled with oxalate and P i . Furthermore, the process of Ca 2+ transport is completely reversible; the outward movement of Ca 2+ is coupled to ATP synthesis from ADP and P i

CDNA CLONING AND PREDICTED PRIMARY STRUCTURE OF SCALLOP SARCOPLASMIC RETICULUM CA2+-ATPASE

Abstract Sarcoplasmic reticulum (SR) Ca2+-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with ∼70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca2+-ATPases is discussed.

Ca2+/Mg2+-Dependent ATPase in Sarcoplasmic Reticulum

Since Hasselbach and Makinose (1961) showed the existence of the Ca2+/Mg2+-dependent ATPase in the membrane of isolated sarcoplasmic reticulum (SR), considerable progress has been made concerning the mechanism of the active transport of Ca2+. The outline of the transport mechanism, which has been obtained mainly from kinetic studies of the Ca2+/Mg2+-dependent ATPase, is described briefly as follows: 1 mole of ATP and 2 moles of Ca2+ bind to 1 mole of the membrane-bound ATPase on the outside of the SR vesicle. The terminal phosphate of ATP is transferred to an aspartyl residue of the enzyme to form a phosphoenzyme. At the same time, Ca2+ is translocated from outside to inside the membrane. The phosphoenzyme is then hydrolyzed in the presence of Mg2+. The entire process of Ca2+ transport can be reversed. When SR vesicles loaded with Ca2+ are reacted with P i in the presence of Mg2+ and EGTA, 1 mole of phosphoenzyme is formed. When ADP is added to the phosphoenzymes, 2 moles of Ca2+ are released accompanying the formation of 1 mole of ATP. These studies have been reviewed in detail by Hasselbach (1979), Inesi (1979), Martonosi (1975), Tada et al. (1978), and Yamamoto et al. (1979).

Substrate‐induced conformational changes in sarcoplasmic reticulum Ca2+‐ATPase probed by surface modification using diethylpyrocarbonate with mass spectrometry

We have identified 15 residues from the surface of sarcoplasmic reticulum Ca2+‐pump ATPase, by mass spectrometry using diethylpyrocarbonate modification. The reactivity of 9 residues remained high under all the conditions. The reactivity of Lys‐515 at the nucleotide site was severely inhibited by ATP, whereas that of Lys‐158 in the A‐domain decreased by one‐half and increased by five‐fold in the presence of Ca2+ and MgF4, respectively. These are well explained by solvent accessibility, pK a and nearby hydrophobicity of the reactive atom on the basis of the atomic structure. However, the reactivity of 4 residues near the interface among A‐, N‐ and P‐domain suggested larger conformational changes of these domains in membrane upon binding of Ca2+ (Lys‐436), ATP (Lys‐158) and MgF4 (His‐5, ‐190, Lys‐436).

The Ca2+-ATPase of the Scallop Sarcoplasmic Reticulum Is of a Cold-adapted Type

At 0 to 20°C, the Ca2+-ATPase activity of the scallop sarcoplasmic reticulum (SR) was observed to be 7–60% of the peak activity at 30°C, while the ATPase activity of the rabbit SR was 0–7% of its peak at 55°C. The relative rabbit ATPase activity (0.7–7.0%) at 7–20°C became higher (6–15 times) and lower (1/4–1/2), respectively, by the solubilization of the rabbit ATPase with a detergent, dodecyloctaethylenglycol monoether, and by the reconstitution of the ATPase with asolectin (soybean lecithin). No activity at 0°C remained irrespective of these treatments. The relative scallop ATPase activity at 0–20°C was, however, scarcely affected by such solubilization and reconstitution. In contrast to the rabbit ATPase, the scallop ATPase seems to be capable of operating independently without the help of the membrane lipid at low temperature.

Irradiation with Ultraviolet Light in the Presence of Vanadate Increases Ca2+ Permeability of the Sarcoplasmic Reticulum Membrane via Ca2+-ATPase

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2 mM EGTA, 10 mM MgCl2, 20% DMSO, and 50 mM PIPES (pH 6.5) at room temperature. In the presence of 100 microM vanadate, the Ca(2+)-uptake activity of SR rapidly decreased and was almost lost in 20 min. The activity was inhibited as a function of vanadate concentration with an apparent Ki of about 20 microM. On the other hand, Ca(2+)-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20 min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 microM vanadate. The loss of the relationship between Ca(2+)-uptake and ATPase reaction was found to be mainly caused by an increase in the Ca2+ permeability of the SR membrane, which was raised by increasing the vanadate concentration or UV irradiation time in a manner similar to that observed for the Ca2+ uptake. No rise in Ca2+ permeability occurred in liposomes reconstituted from SR lipid when they were irradiated with UV in the presence of 100 microM vanadate. When the vanadate-UV-treated SR was allowed to react with fluoral-P (4-amino-3-penten-2-one), an indicator of aldehyde, and the membrane proteins were separated by HPLC in the presence of SDS, the fluorescent probe was found to be closely associated with the Ca(2+)-ATPase fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

OLIGOMERIC FORM OF THE Ca2+, Mg2+-DEPENDENT ATPase IN SARCOPLASMIC RETICULUM

Ca 2+ , Mg 2+ -dependent ATPase is a major component of the sarcoplasmic reticulum (SR) membrane that accounts for more than 70% of the total protein. It serves as a translocator for Ca 2+ as well as an energy transducer. However, the manner in which the movement of the enzyme molecule is coupled to the Ca 2+ translocation across the membrane and associated with ATP hydrolysis is not clearly understood. To better understand the molecular basis of the active transport of Ca 2+ , information is needed concerning the structural aspects of the Ca 2+ , Mg 2+ -dependent ATPase molecules in the sarcoplasmic reticulum membrane. One of the most important findings in the kinetic studies on the Ca 2+ , Mg 2+ -dependent ATPase reaction in the presence of C 12 E 8 is that the effect of C 12 E 8 on the catalytic function of the sarcoplasmic reticulum ATPase is entirely reversible.