Giuseppe Inesi

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

Abstract A transition in the temperature dependences of Ca 2+ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca 2+ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition. Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca 2+ accumulation and ATPase activity are uncoupled, and Ca 2+ accumulation is inhibited, while ATPase activity and passive Ca 2+ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.

A study of the phosphorylated intermediate of sarcoplasmic reticulum atpase

Abstract Incorporation of ATP terminal phosphate into sarcoplasmic reticulum (SR) occurs very rapidly on ice. Ca 2+ is a requirement and half-maximal activation is obtained in the presence of 1 × 10 −7 , m Ca 2+ . The activating Ca 2+ concentrations are identical for phosphate incorporation and ATPase, while excess Ca 2+ only inhibits ATPase. The rate of enzymatic degradation of the 32 P-membrane complex, in the presence of Mg 2+ , is comparable to that of ATP hydrolysis. Mg ++ accelerates the P- membrane complex turnover. The pH stability of the phosphorylated species is different in membranes denatured at acid pH (trichloroacetic acid), as opposed to membranes denatured at neutral pH (Salyrgan, acetone) and then exposed to various pH values. ATPase activity and steady-state levels of 32 P-membrane complex are reduced when SR is incubated in the presence of hydroxylamine. However, SR incubated with hydroxylamine in optimal conditions for formation of P-membrane complex, regains full activity after washings. This lack of permanent inactivation suggests that hydroxylamine does not form a stable hydroxamate at the active site. Degradation of trichloroacetic acid-treated 32 P-membrane complex is accelerated by hydroxylamine. On the other hand, 0 -( 14 C) methylhydroxylamine is bound by trichloroacetic acid-treated SR, independent of its previous incubation in the presence or in the absence of ATP. This indicates that the hydroxylamine effect may not be related to a specific reaction with an acylphosphate. Solubilization of 32 P-membrane complex with Triton X 100 at acid pH, and gel chromatography, yield phosphorylated particles with 60–80% of the original specific activity. On the other hand, particles solubilized with deoxycholate at neutral pH retain only traces of 32 P. A mechanism is proposed for SR ATPase, in which Ca 2+ binding to the membrane (a) occurs as a consequence of site modification induced by ATP binding, and (b) activates phosphoryl transfer and formation of a phosphorylated intermediate.

Phase changes in the lipid moieties of sarcoplasmic reticulum membranes induced by temperature and protein conformation changes

Lipid alkyl regions in sarcoplasmic membranes were probed with hydrophobic spin labels in the temperature range between 5 and 50 °C. Results indicate that biochemically active preparations undergo transitions at 22 and 40 °C. The first one is associated with a melt of lipid alkyl chains and does not depend on membrane proteins. Evidence is presented that the second transition reflects hydrophobic lipid-protein interactions and the effect of protein conformational changes on the dynamic state of membrane lipids. In addition, proteolytic digestion of surface granular structures does not affect the behavior of lipid-soluble hydrophobic spin labels or the detected lipid-protein interactions. The partitioning of an amphiphilic spin label between polar and apolar regions is affected, however, indicating that some surface proteins interact with the lipids via polar forces.

The involvement of sarcotubular membranes in genetic muscular dystrophy.

Microsomal preparations from breast muscle of normal and dystrophic chickens are characterized with regard to ultrastructural features, protein composition, Ca2+ transport and ATPase activity. Dystrophic muscle yields a greater microsomal dry weight, with a reduced protein to lipid ratio. This is related to the presence of a considerable number of low density microsomes, in addition to seemingly normal microsomes. The low density microsomes display a reduced number of protein particles on freeze fracture faces. Electrophoretic analysis reveals nearly identical patterns in normal and dystrophic microsomes. Furthermore, normal and dystrophic microsomes sustain equal rates of Ca2+ transport and ATPase, demonstrating an identical protein specific activity. However, the dystrophic microsomes have a lower capacity to retain transported Ca2+. The high yield of low density microsomes with reduced capacity for Ca2+ uptake is attributed to the presence of membranes proliferated in the junctional and tubular sarcomere regions of the dystrophic muscle. It is suggested that proliferation of such membranes accounts for the altered excitation-contraction coupling and cable properties of genetically dystrophic muscle.

Detection of an initial burst of Ca2+ translocation in sarcoplasmic reticulum

Abstract Rapid quench methods were used to determine Ca2+ uptake, ATPase phosphorylation and Pi production in the transient state of Sarcoplasmic Reticulum. It was found that within 20 milliseconds of the addition of ATP maximal levels of phosphorylated enzyme intermediate are reached and an initial burst of Ca2+ uptake is completed. This burst, kinetically distinct from the following transport activity, is related to the phosphorylated intermediate with a molar ratio of two.

Mg2+ and Mn2+ modulation of Ca2+ transport and ATPase activity in sarcoplasmic reticulum vesicles

Abstract Kinetic experimentation was used to characterize the Mg 2+ and Mn 2+ modulation of Ca 2+ transport and ATPase activity in sarcoplasmic reticulum vesicles. In addition to its participation in the ATP·Mg complex as substrate for the ATPase, Mg 2+ is an activator of phosphoenzyme progression to hydrolylic cleavage. It is shown that this activation is due to Mg 2+ occupancy of an allosteric site easily accessible on the outer surface of the vesicles, rather than to participation in an antiport mechanism. The Mg 2+ site is distinct from the Ca 2+ binding sites which are involved in activation of enzyme phosphorylation by ATP, and Ca 2+ translocation. The role of Mg 2+ is quite specific, inasmuch as phosphoenzyme decay is much slower if the Mg 2+ allosteric site is occupied by Ca 2+ . Conversely, competive occupancy of the Ca 2+ sites by Mg 2+ does not permit enzyme phosphorylation by ATP. Intermediate characteristics between Mg 2+ and Ca 2+ are displayed by Mn 2+ which is well able to stimulate phosphoenzyme cleavage by occupancy of the Mg 2+ allosteric site, and is also able (although at much slower rates) to activate enzyme phosphorylation, and undergo active transport by occupancy of the Ca 2+ sites.

ATP dependent conformational change in “spin labelled” sarcoplasmic reticulum☆

Abstract Spin labelling of membrane vesicles was obtained by reacting fragmented sarcoplasmic reticulum with nitroxide-iodoacetamide. The EPR spectra of the reacted label showed two components; corresponding to a “weakly immobilized” and a “strongly immobilized” signal. A slowly reversible change of the EPR spectra was obtained on addition of ATP. The presence of Mg++ and Ca++ did not influence the appearance of such a change, but prompted a more rapid reversal. An effect similar to that of ATP was obtained with ADP, ITP and by increasing the pH of the membrane suspension above 9.1. Solubilization of the membrane with deoxycholate and denaturation with guanidine markedly changed the EPR spectra of the spin labels. The effect of ATP suggests a conformational change due to simple binding of ATP to the membrane.

The ultrastructure of membrane alterations of enzymatically dissociated cardiac myocytes

Enzymic dissociation of cardiac myocytes results in a homogeneous population of cylindrical cells, if the Ca2+ concentration in the medium is rigorously controlled. In the presence of 0.5 to 1 μ m Ca2+ the myocytes undergo cyclic contraction, even though no resting or action potentials can be recorded. The automatic behavior of the myocytes is highly sensitive to the Ca2+ concentration in the medium, but is not affected by tetrodotoxin or changes in K+ and Na+ concentrations. Structural studies by scanning and transmission electron microscopy on whole cells, sections, and freeze-fracture preparations reveal a loss of basement membrane, but a normal continuity of the plasma membrane over the entire surface of cylindrical myocytes, consistent with the exclusion of dyes of approximately 900 MW. The dissociation results in separation of the intercalated discs with either uncoupling or tearing of gap junctions. Tearing results in both membranes of the gap junction remaining with one of the cells and resealing of the torn membranes appears to occur readily. Twenty to 30% of gap junctions, however, are left with one cytoplasmic face exposed to the medium, likely providing channels for entry of electrolytes. This is consistent with the observed electrochemical shunt, the permeability to Ca2+ and the pattern of contractile activation originating at the polar ends of the myocytes where the exposed gap junctions are located. Freeze-fracture studies reveal normal ultrastructure detail, with the exception of lower and altered distribution of protein particles in the plane of nonjunctional myolemma. Sarcoplasmic reticulum and sarcomeres appear intact in all details, consistent with a normal involvement of these structures in contractile activation of dissociated myocytes.

An ultrastructural study of calcium induced degenerative changes in dissociated heart cells

Abstract Dissociated myocytes were prepared by enzymatic perfusion of adult rabbit hearts, and fixed following exposure to various concentrations of Ca2+. Only slight ultrastructural changes were noted in myocytes which were maintained in relaxation [(Ca2+) m ] or in cyclic contractile activity [(Ca2+) ≅ 0.4 μ m ]. In these cells, slight swelling permitted clear demonstration of interesting ultrastructural details such as tight connections between Z bands and sarcolemma, attachment of filaments to these structures, and relationships of sarcoplasmic reticulum and T tubules. When the Ca2+ concentration was raised above 1.0 μ m , sarcomeres exhibited extreme and irreversible shortening, followed by myofilament disorganization, disappearance of Z lines, aggregation of most thick and some thin filaments forming a central mass, and outward displacement of organelles. Under these conditions numerous filaments were attached randomly to the sarcolemma and to intracellular membranes, demonstrating their inherent capacity to bind to membranous structures. The ultrastructural alterations observed in our experiments are similar to those described for the “calcium paradox”, which is likely to be a common pathogenic mechanism of myocardial cell damage.

Ca2+ transport and assembly of protein particles in sarcoplasmic membranes isolated from normal and dystrophic muscle

Vesicles of fragmented sarcoplasmic reticulum (SR) membranes have one major activity: Ca2’ uptake coupled to ATP hydrolysis. Their simplicity make them particularly suitable for studying structurefunction relationships. In this paper we compare the structural and the biochemical properties of normal and dystrophic SR obtained from chicken pectoralis muscle. We show that electrophoretic patterns of solubilized protein and specific ATPase activity are very similar in normal and dystrophic SR, whereas the density of protein particles revealed by freeze fracture and the occurrence of fracture faces containing particles is lower for dystrophic microsomes. Associated with this we find a reduction of ATP dependent calcium uptake in dystrophic SR.