Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains

Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.     

Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.     

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica on Potato Tuber Tissue

The elicitation of pathogenesis on potato tuber slices by 6 strains of Erwinia carotovora ssp. atroseptica was investigated by neutral red vital staining and has been compared with bacterial growth rate, penetration ability, enzyme production and enzyme spectrum. The induction of enzyme synthesis (particularly, of the extracellular polygalacturonase) elicites the rot attack on tuber tissue and this requires a sufficient bacterial density. Due to wound healing, the inductors of enzyme production are removed, and after 48 h enzymes do not attack tuber tissue any more. Therefore, growth rate and penetration ability (to get the necessary bacterial density and inductor substances) may limit virulence. A similar influence of enzyme production and enzyme spectrum of the strains on the virulence was not detected.     

Autoradiographische Untersuchung über tageszeitliche Schwankungen des H-3-Index und des Mitose-Index bei Zellarten der ausgewachsenen Maus,des Ratten-Fetus sowie bei Ascites-Tumorzellen

Zusammenfassung Für 10 Zellarten der normalen Maus, für 5 fetale Zellarten der Ratte (20. Tag der Gravidität) und für 3 Aszites-Tumoren wurde das Auftreten von tageszeitlichen Schwankungen des H-3-Index und des Mitose-Index untersucht. Die Tötung der Tiere erfolgte jeweils 40 min nach Injektion von H-3-Thymidin. Von den Organen wurden Autoradiogramme hergestellt.Bei einer Reihe von Zellarten des erwachsenen Tieres wurden neben den bekannten tageszeitlichen Schwankungen des Mitose-Index auch Schwankungen des H-3-Index gefunden.Bei allen Zellarten mit Schwankungen des Mitose-Index wurden ohne Ausnahme auch solche des H-3-Index beobachtet. Demgegenüber zeigten bei anderen Zellarten beide Indices keine Schwankungen.Bei den fetalen Zellarten und den Ascites-Tumoren wurden weder tageszeitliche Schwankungen des H-3-Index noch des Mitose-Index gefunden.Die tageszeitlichen Schwankungen des H-3-Index werden als — bei einer bestimmten Tageszeit auftretende — partielle Synchronisationen der Zellen gedeutet. Aus den gemessenen Kurven des H-3-Index wird der tageszeitliche Verlauf der Eintritts-Rate der Zellen in die S-Phase berechnet (Signalkurve).Es wird diskutiert, ob die tageszeitlichen Schwankungen des Mitose-Index als ein selbständiger Prozeß oder als eine notwendige Folge der H-3-Index-Schwankungen zu deuten sind.

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Summary Diurnal fluctuations of the H-3-index and the mitotic index were investigated in 10 different cell types of normal mice, in 5 fetal cell types (rats, 20th day of pregnancy) and in 3 ascites tumors. The animals were sacrified 40 minutes after injection of H-3-thymidine. Autoradiographs were prepared from various organs.Diurnal fluctuations of the H-3-index were observed in a number of cell types of the adult animal, aside from the diurnal fluctuations of the mitotic index known already. These H-3-index fluctuations were found without exception in all cell types in which diurnal fluctuations of the mitotic index were observed. In other tissues diurnal fluctuations could not be demonstrated with both indices. Diurnal fluctuations of the H-3- and the mitotic index were also not present in the fetal cells and in the ascites tumours.The diurnal fluctuations of the H-3-index are regarded as partial synchronisations of cells which occur at a certain time of day. The daily time course of the rate of entry of cells into S-phase is calculated from the H-3-index curves measured (signal curve).It is discussed whether the diurnal fluctuations of the mitotic index are to be regarded as an autonomous process or as a necessary consequence of the H-3-index fluctuations.

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Wir möchten Herrn Dr. W. Erb für Mithilfe bei der Auswertung der Versuche, der Fa. Dr. Madaus & Co., Köln-Merheim, für die Überlassung der Versuchstiere herzlich danken. Weiterhin danken wir Herrn Dr. L. Révész, Karolinska Institut, Stockholm, für die Überlassung des tetraploiden Ehrlich-Ascites-Tumors. Die Arbeit wurde durch Mittel der Gesellschaft zur Bekämpfung der Krebskrankheiten in Nordrhein-Westfalen und des Bundesministeriums für Wissenschaftliche Forschung unterstützt.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum-Free Medium

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na⁺,K⁺-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm² in the presence of serum to 1,500 Ω· cm² after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na⁺,K⁺-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.