Claus Heilmann

Identification of the Zn2+ binding region in calreticulin.

Calreticulin binds Zn2+ with the relatively high affinity/low capacity. To determine the location of the Zn2+ binding site in calreticulin different domains of the protein were expressed in E. coli, using the glutathione S‐transferase fusion protein system, and their Zn2+‐dependent interaction with Zn2+‐IDA‐agarose were determined. Three distinct domains were used in this study: the N + P‐domain (the first 290 residues); the N‐domain (residues 1–182) and the proline‐rich P‐domain (residues 180–273). The N + P‐domain bound to the Zn2+‐IDA‐agarose and were eluted with an increasing concentration of imidazole. The N‐domain also bound 65Zn2+ as measured by the overlay method. The P‐domain did not interact with the Zn2+‐IDA‐agarose and it did not bind any detectable amount of Zn2+. Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn2+ but they were modified by diethyl pyrocarbonate in the absence of Zn2+ suggesting that these residues may be involved in Zn2+ binding to calreticulin. We conclude that Zn2+ binding sites in calreticulin are localized to the N‐domain of the protein, region that is not involved in Ca2+ binding to calreticulin.

Correlation between ultrastructural and functional changes in sarcoplasmic reticulum during chronic stimulation of fast muscle

SummaryChronic indirect stimulation of fast twitch rabbit muscle (tibialis anterior and extensor digitorum longus) with a frequency of 10 Hz induced a progressive transformation of the sarcoplasmic reticulum (SR). Ultrastructural changes as studied by electron microscopy of freeze-fractured vesicles consisted in a decrease of intramembranous particles of the concave (A) face and an increase of particles in the convex (B) face. The asymmetry of the membrane proved to be lowered. Changes in the particle density of theA face were mainly confined to the 7–9 nm particles. Electrophoretic analyses revealed a decrease in the 115,000-Mr Ca2+ transport ATPase. The reduced density of the 7–9 nm particles correlated well with decreased activities in Ca2+-dependent ATPase as well as with decreases in initial and maximum Ca2+ uptake.

Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes

We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructural localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium binding protein calsequestrin by immunoabsorption on isolated rat liver hsp60. As shown by partial N-terminal amino acid sequence analysis of immunoprecipitated proteins AB 121 contained reactivities against hsp60, calsequestrin and the glycoprotein fetuin. In rat heart AB 121 recognized calsequestrin and hsp60. In human and rat liver the only reacting protein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody was used for immuno-gold electron microscopical localization of hsp60: in myocardiocytes and hepatocytes of the rat strong labelling was obtained exclusively in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visualized by immuno-gold electron microscopy offers the possibility to study the expression of this protein in the liver and in other organs. Possible clinical applications of these studies are discussed, since hsp60 could be a target antigen of autoantibodies in diseases such as autoimmune hepatitis, primary sclerosing cholangitis or primary biliary cirrhosis.

The phosphoprotein intermediate of a Ca2+ transport ATPase in rat liver endoplasmic reticulum

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca2+ transport which is mediated by a (Ca2+ + Mg2+)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-Mr in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-Mr phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca2+ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.

Characterization of cardiac microsomes from spontaneously hypertonic rats

Capacity of Ca2+ sequestration was found to be significantly lowered in microsomal preparations of hearts from spontaneously hypertonic rats. A decrease to 40% of the control level was found for basal and extra ATPase. A similar reduction existed in initial and total Ca2+ uptake. These findings are correlated with a lower concentration of the Ca2+ transport ATPase in SDS gel electrophoresis, a lower density of the 7–9 nm intramembraneous particles and higher half-lives of phosphoprotein. Altered contractility of hypertrophied myocardium may thus be partially explained by the dysfunction of the Ca2+ sequestering system.

Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties

It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins.

Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates.

Microsomal fractions, highly enriched with endoplasmic reticulum of rat and human liver exhibit Ca2+ uptake catalyzed by a Ca2+-pumping ATPase. The mechanism of Ca2+-translocation involves: (i) reversible Ca2+-dependent formation of an acyl-phosphoenzyme intermediate (Mr 116,000 to 118,000) with bound Ca2+, which in the reversed reaction can transphosphorylate its Pi to ADP to re-synthesize ATP; (ii) reversible transition of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, not further reactive to ADP; (iii) hydrolytic cleavage, stimulated by Mg2+, K+, and ATP of the ADP-unreactive phosphoenzyme with liberation of Pi. By analogy to a mechanism proposed for the Ca2+ pump of sarcoplasmic reticulum, the translocation of Ca2+ to and dissociation from the inner side of the membrane is suggested to occur by a conformational change, coupled with a decrease in Ca2+-affinity of the phosphoenzyme during its transition into the ADP-unreactive isomer. With CaATP as the effective substrate the reactions proceed normally but at a considerably slower rate.

Characterization of cardiac microsomes from spontaneously hypertonic rats@@@Charakterisierung der Mikrosomenfraktion von Herzen spontan hypertonischer Ratten

SummaryCapacity of Ca2+ sequestration was found to be significantly lowered in microsomal preparations of hearts from spontaneously hypertonic rats. A decrease to 40% of the control level was found for basal and extra ATPase. A similar reduction existed in initial and total Ca2+ uptake. These findings are correlated with a lower concentration of the Ca2+ transport ATPase in SDS gel electrophoresis, a lower density of the 7–9 nm intramembraneous particles and higher half-lives of phosphoprotein. Altered contractility of hypertrophied myocardium may thus be partially explained by the dysfunction of the Ca2+ sequestering system.ZusammenfassungMikrosomale Präparationen aus Herzen von spontan hypertonischen Ratten zeigten eine signifikant herabgesetzte Kapazität zur Ca-Aufnahme. Die Aktivitäten von basaler und Extra-ATPase waren gegenüber den Kontrollen auf 40% erniedrigt. Initiale und gesamte Ca-Aufnahme waren in ähnlichem Ausmaß herabgesetzt. Diese Befunde lassen sich mit einer erniedrigten Konzentration der Ca-Transport-ATPase in SDS-Gelelektrophoresen sowie einer niedrigeren Dichte der 7–9-nm-Partikel in Membrangefrierbrüchen und schließlich den erhöhten Werten der für Phosphoproteine ermittelten Halbwertszeiten korrelieren. Die veränderte Kontraktilität des hypertrophierten Myokards läßt sich somit möglicherweise teilweise auf eine gestörte Funktion der mikrosomalen Membranen zurückführen.