The structure and function of alpha-actinin.
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Actinin↗
Biomedical subjects
V Ohanian
Publications and source records attributed to V Ohanian.
Prenatal diagnosis of hereditary red cell membrane defect.
The red cells of a severely anaemic 2-year-old child of a white British family showed high haemolytic fragility with poikilocytosis. The cells showed markedly impaired thermal stability. The mother was phenotypically normal, but the father's red cells showed mild elliptocytosis. The spectrin from the latter, extracted at low temperature, was 30% dimeric (cf. 5-10% in normals). Tryptic digests of the spectrin from both father and daughter showed a reduction in the fragment of 80,000 molecular weight, derived from the terminus of the alpha-chain, and the elevation of a fragment of molecular weight 46,000, as well as one of 53,000. These characteristics and the autosomal recessive inheritance lead to a diagnosis of type II hereditary pyropoikilocytosis, so far reported only in two black American families (Lawler et al, 1983). The spectrin from the father was examined with respect to thermal conformational stability, and was found to be normal. The spectrin from the cells of the daughter gave evidence of the presence of oxidative (disulphide) cross-links, as well as of extensive noncovalent aggregation. Blood was obtained from the umbilical cord vein of the 19-week fetus of the pregnant mother: 250 microliters of blood was used for preparation of red cell membranes for SDS-gel electrophoresis and for extraction of spectrin. Analysis of the spectrin by gel electrophoresis in the native state revealed that the proportion of dimer was within the normal range, and the fetus therefore did not possess the hereditary pyropoikilocytosis phenotype. It is suggested that the procedures described could be generally applied to the prenatal identification of phenotypes associated with severe haemolytic anaemias.
Anemia, Hemolytic, Congenital↗
A case of elliptocytosis associated with a truncated spectrin chain.
A case of haemolytic anaemia with elliptocytosis is described, in which a large part of the smaller (beta) subunit of the spectrin is truncated, and has an apparent molecular weight of about 214 000 compared with about 230 000 for the normal chain. It is shown that this is not a product of adventitious proteolysis during lysis or extraction. At the same time about 35% of the total spectrin in the cells is liberated from the membrane as the dimer (which is present in normal cells to the extent of less than 10%). The truncated (beta') chain appears exclusively in this dimer fraction. The beta'-chain is incapable of phosphorylation by the endogenous cAMP-independent membrane kinase, and it may be inferred that the deleted segment of the chain contains both the spectrin self-association site and the residues normally phosphorylated. The alpha beta'-dimer is active with respect to participation in a ternary complex with its partnering proteins in the membrane cytoskeleton, F-actin and 4.1, confirming that the phosphorylation sites are not involved in the primary interaction with the other cytoskeletal proteins at the network junctions. The spectrin alpha-chain generates the terminal tryptic fragment of molecular weight 80 000 characteristic of normal spectrin, rather than the 74 000 molecular weight peptide derived from the alpha-chain in cases of hereditary elliptocytosis and pyropoikilocytosis, associated with anomalous self-association of spectrin dimer. Membrane cytoskeletons, extracted from the patient's red cells, undergo normal gelation on incubation with cAMP-independent kinase and ATP, and thus do not resemble those derived from hereditary spherocytosis cells. The properties of the anomalous spectrin resemble in most respects that described in a French family by Dhermy et al (1982).
Adolescent↗
Preparation of red-cell-membrane cytoskeletal constituents and characterisation of protein 4.1.
A new and rapid method is described for the preparation of protein 4.1, the protein which modulates the interaction between spectrin and actin in the membrane cytoskeleton of the red cell. The method is based on the dissociation of purified membrane cytoskeletons in concentrated Tris at neutral pH, followed by gel filtration in the same medium. This procedure also yields spectrin and actin, as well as the fourth cytoskeletal constituent, protein 4.9, in relatively pure form, and ankyrin. Protein 4.1 is monomeric under our conditions of solvent and protein concentration, with a relative molecular mass, as determined from sedimentation equilibrium, of about 78 000; its sedimentation coefficient and Stokes' radius are those of a globular, though somewhat asymmetric or flexible molecule. It forms a strong complex with F-actin and spectrin. Protein 4.9 is also recovered in active form, and will bind strongly to F-actin.
Actins↗
Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1.
Spectrin dimers interact weakly with F-actin under physiological solvent conditions (with an association constant of about 5 X 10(3) M-1 at 20 degrees C). In the presence of the membrane skeletal constituent, protein 4.1, strong binding is observed; an analysis of the profiles for formation of a ternary complex leads to an association constant of about 1 X 10(12) M-2. This association becomes weaker at low ionic strength, whereas the opposite applies to the spectrin-actin interaction. The stability of the ternary complex is maximal at physiological ionic strength and somewhat above. The effect of temperature in the range 0-20 degrees C on the formation of the ternary complex is small, whereas the spectrin-actin interaction almost vanishes at low temperature. There is no detectable calcium sensitivity in either the binary or the ternary system within the limits of precision of our assay. The ternary complex resembles the natural system in the membrane in that the actin is resistant to dissociation and unavailable in the deoxyribonuclease assay; after selective proteolytic destruction of spectrin and 4.1, all the actin becomes available. In the absence of 4.1, spectrin dimers do not measurably protect the actin against dissociation.
Actins↗
Spectrin and protein 4.1 as an actin filament capping complex.
Spectrin and protein 4.1, when added to G- or F-actin, cause the formation of short filaments, as judged by the appearance of powerful nucleating activity for G-actin polymerisation. F-Actin filaments are rapidly fragmented under physiological solvent conditions. The effect of cytochalasin E on the polymerisation reaction and the extent of reduction in the critical monomer concentration of actin when spectrin and 4.1 are added suggest that these proteins form a capping system for the more slowly growing, or 'pointed' ends of actin filaments. The interaction is not affected by calcium or by 4.9, the remaining constituent of the purified red cell membrane cytoskeleton.
Actins↗
Properties of brain spectrin (fodrin).
Fodrin, a protein from bovine brain, immunologically related to spectrin, is shown, unlike some other proteins of generally similar appearance in the electron microscope, to resemble spectrin closely in its most distinctive structural characteristic, the very high alpha-helix content. Like spectrin, it is also insoluble below pH 5. One of the subunits only is phosphorylated by the cAMP-independent red cell membrane kinase, that phosphorylates the smaller subunit of spectrin. Fodrin also forms a ternary complex with F-actin and the third constituent of the red cell membranes cytoskeleton, protein 4.1. In the presence of 4.1 the interaction between fodrin and F-actin is enhanced. It is surmised that fodrin plays an analogous functional role in neuronal cells to that of spectrin in the red cell.
Actins↗
In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte.
The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.
Actins↗
Endothelin-1 stimulates hydrolysis of phosphatidylcholine by phospholipases C and D in intact rat mesenteric arteries.
A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [3H] or [14C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [3H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [3H]PA levels, but did not alter the time course of formation. In addition, [14C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [3H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 microg/ml, completely abolished the ET-1-induced increase in [14C]DAG without affecting the increase in [14C]PA, PLD activity or the contractile response. In [14C]choline-labelled vessels, [14C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca2+ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 microM) abolished ET-1-induced [3H]PA, [3H]PEt and [14C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.
Animals↗