Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.     

Preparation of passive bilayer liposomes

In studies of in-membrane molecular interactions, need may arise for a matrix that cannot itself interact, except hydrophobically, with the reactants. Such a bilayer matrix should, ideally, consist of only a hydrophobic zone without ionic outer layers and without hydrogen belts (the membrane strata containing CO and OH groups). However, because of the necessity of anchoring the bilayer to its aqueous surroundings, there must be polar substituents. Hydrophilic ether groups in the form of polyoxyethylenes can provide nearly sufficient anchoring and yet not confer unwanted reactivity to the membrane since they are only very weak H-bond acceptors. The stability of the bilayer is ensured by the presence of a few percent of an amphiphile (which may be the substrate to be studied, e.g. a phospholipid) or by a free polyethylene hydroxy group far remote from the original hydrogen belt region. Our most impermeable liposomes consisted of O-methylcholesterol/O-methoxyethoxyethoxyethylcholesterol; the most readily prepared liposomes were made from O-methylcholesterol and hydroxy(ethoxy)4dodecane (Brij 30) or Triton.     

Phosphatidylcholine and cholesterol inhibit phosphatidate-mediated calcium traversal of liposomal bilayers

Rates of phosphatidic acid- (PA-) mediated Ca2+-traversal are maximal in 'passive bilayers' void of lipid CO and OH groups: dietherphosphatidylcholine (diether-PC) or OH-blocked cholesterol liposomes. Phosphatidylcholine (PC) as bilayer matrix causes 99% inhibition, while 45 mol% cholesterol in passive bilayers inhibits by about 70%. Possibly, the absence of CO and OH groups causes a dehydration of the 'hydrogen belts', i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine) and thereby facilitates the formation of dehydrated Ca(PA)2, the ionophoric vehicle; or (our preferred explanation) PC engages in a (non-ionophoric) Ca(PA X PC) complex and thus reduces the concentration of the ionophore, while cholesterol competes with Ca2+ for the CO groups of phosphatidic acid by hydrogen-bonding. The Ca2+-traversal rates realized in bilayers with modified hydrogen belts lend support to the speculation that a Ca(PA)2 ferry may be of physiological importance, e.g., in membranes (such as myelin) containing much ether phospholipid (plasmalogen); and that Ca2+-membrane association and traversal may be controlled by the composition of the hydrogen belts.     

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+.     

Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.     

Molecular interactions in the hydrogen belts of membranes. Glucose-6-phosphatase, lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2- deoxylysophosphatidylcholine , is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.     

Polyphosphoinositide mono- an diphosphoesterases of three subfractions of rat brain myelin

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the low density, medium density, and heavy density myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the heavy density over the low density myelin fraction. These results show that the hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Rapid Incorporation In Vivo of Intracerebrally Injected 32Pi into Polyphosphoinositides of Three Subfractions of Rat Brain Myelin

Abstract: At intervals ranging from 1 to 10 min after injection of ³²Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho-inositides (PPI) was then determined. There was rapid incorporation of ³²Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated ³²Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos-phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged ³²Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.