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PubMed1984

[Molecular interactions of membrane proteins and erythrocyte deformability].

The structural and functional properties of the erythrocytic membrane constitute one of the essential elements of the red cell deformability. They intervene not only in the flexibility of the membrane, but also in the surface/volume relation and, through transmembrane exchanges, in the internal viscosity of the red cells. These properties depend essentially on the molecular composition of the elements which constitute the membrane, and on their interactions. The shape of the red cell and the flexibility of its membrane depend, to a great extent, on the membrane skeleton, whose main components are spectrin, actin, and protein 4.1. The spectrin basic molecule is a heterodimer, but there occur interactions between dimers in vitro as well as in vivo, which lead to the formation of tetrameric and oligomeric structures of higher complexity. Disturbances of these interactions, such as have been observed in pathological cases, lead to an instability of the membrane, a loss of membrane fragments, and a decrease in the surface/volume relation, with, as a consequence, a reduced deformability. The stability of the membrane skeleton also depends on the interactions between spectrin and protein 4.1. These interactions occur through a binding site on the beta chain of spectrin apparently close to actin and calmodulin binding sites. Other interactions occur between the hydrophobic segment of spectrin and membrane lipids. The cytoskeleton is bound to the transmembrane proteins: by ankyrin to the internal segment of protein band 3, and by protein 4.1 to a glycoprotein named glycoconnectin. There seems to exist other, more direct, lower affinity bindings between the cytoskeleton on the one hand, and band 3 and glycophorin transmembrane proteins on the other hand, whose lateral mobilities are modified when the structure of the skeleton is perturbed. The membrane proteins, which are in contact with the cytosol, interact with the cytosolic proteins, in particular with certain enzymes involved in anaerobic glycolysis and with hemoglobin. The Hb-membrane interactions are complex and involve different binding types with various affinities, as through band 3 protein, phospholipids, and glycophorin. These bindings are particularly important in the case of certain abnormal hemoglobins such as S and C. If the existence of molecular interactions inside the membrane and their sites are fairly well known, the mechanisms which control or influence such interactions remain unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1981

Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system.

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.

2,3-Diphosphoglycerate↗
PubMed1984

Identification of the hereditary pyropoikilocytosis carrier state.

We evaluated the hematologic, rheologic, and biochemical features of erythrocytes obtained from 10 relatives of a 5-yr-old black female with hereditary pyropoikilocytosis (HPP) and severe hemolytic anemia. Erythrocyte morphology was normal in the father and five other relatives, but ghost mechanical fragility and drug-induced red cell endocytosis were increased, as was the percentage of spectrin dimers noted on 3.2% nondenaturing PAGE of spectrin extracts. Identical changes were also noted in the mother and her sister, whose erythrocytes were elliptocytic and exhibited morphological changes upon heating to 45 degrees-48 degrees C (normal 49 degrees). The two other family members were normal in every respect. SDS-PAGE analysis of membrane proteins demonstrated diminished amounts of spectrin in HPP erythrocytes, but was normal in other family members. A diffuse band (mol wt 575,000-665,000), composed entirely of spectrin, was apparent adjacent to the dimer region on nondenaturing PAGE of spectrin extracts from the propositus, mother, and aunt. In this family, HPP appears to have resulted from compound heterozygosity for two distinct genetic abnormalities (reflected by the differences between elliptocytic and nonelliptocytic carriers). Although the membrane abnormalities in carriers did not result in hemolytic anemia, they were of sufficient magnitude to allow the detection of the carrier state.

Adult↗
PubMed1979

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.

Acetylcholinesterase↗
PubMed1993

Muscle fibre degeneration in distal myopathy (Welander)--ultrastructure related to immunohistochemical observations on cytoskeletal proteins and Leu-19 antigen.

In seven patients with long-standing and six patients with early symptoms of Welander distal myopathy (WDM), monoclonal antibodies directed against such cytoskeletal proteins as dystrophin, spectrin and desmin and against Leu-19, a myoblast and satellite cell related antigen, were applied to muscle biopsies from the anterior tibial and soleus muscles. In addition, ultrastructural studies were carried out on biopsies from the soleus muscle. In muscle fibres from patients with early symptoms there was normal immunostaining for dystrophin, spectrin, desmin and Leu-19. In the patients with long-standing symptoms, there was also a normal expression of dystrophin, and a normal staining for spectrin and desmin was found in normal sized muscle fibres. Occasionally normal sized muscle fibres showed staining for Leu-19. Increased staining for spectrin and desmin and a strong Leu-19 staining was seen in normal sized muscle fibres with rimmed vacuoles and in atrophic fibres. Increased staining for spectrin, desmin and Leu-19 has been described in denervated muscle fibres and, thus, the present findings may support earlier findings of a neurogenic component in Welander distal myopathy. In the soleus muscle, ultrastructural muscle fibre abnormalities conformed to those in the anterior tibial muscle. Many rimmed vacuoles were observed which corresponded, at the ultrastructural level, to autophagic vacuoles. Intranuclear and cytoplasmic filamentous inclusions of the same shape and diameter as in inclusion body myositis were observed.

Adult↗
PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Analysis of Variance↗
PubMed1987

A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract.

The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.

Animals↗
PubMed1992

Different distributions of dystrophin and related proteins at nerve-muscle junctions.

The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.

Animals↗
PubMed1990

UV-induced protein alterations and lipid oxidation in erythrocyte membranes.

Certain ultraviolet radiation-induced effects in skin may result from primary photochemical alterations in cell membranes. We have studied isolated erythrocyte membranes in order to determine the UV-fluence and wavelength dependence for protein alterations and lipid oxidation. Protein crosslinking was detected as high molecular weight protein (greater than 200,000 DA) on polyacrylamide/agarose gel electrophoresis. Spectrin decreased more rapidly than the other membrane proteins upon exposure to lambda = 250-380 nm radiation. Nitrogen-purging inhibited the UV-induced decrease in spectrin by 60% and decreased crosslinking to an even greater degree. The decrease in spectrin was not inhibited by superoxide dismutase, catalase, or sodium azide. Radiation at 280 nm was most effective for spectrin loss, 265 and 297 nm were less effective and 254 and 313 nm were not effective. Prior irradiation at 280 nm did not sensitize the membranes to subsequent irradiation at 313 nm indicating that photodecomposition products of tryptophan are not involved. Lipid photooxidation was measured with the thiobarbituric acid assay and was induced at higher fluences of UV radiations than those required for loss of spectrin. These results indicate that the major effects of UV radiation on cell membranes are alterations of proteins and suggest that tryptophan is the major chromophore for these alterations.

Erythrocyte Membrane↗
PubMed1993

[Presence and localization of proteins immunologically related to membrane skeletal proteins in human skin].

Recent biochemical studies have shown that spectrin, protein 4.1, and actin form a skeletal protein network that underlines the inner surface of the erythrocyte membrane. The skeleton is a flexible structure that appears to be important in maintaining the shape and mechanical properties of the erythrocyte. Recent studies indicate that immunoanalogues of erythrocyte membrane skeletal proteins and ankyrin (protein 2.1) have been found in a wide variety of non-erythroid tissues. In the present study, in order to examine if human skin contains membrane skeletal proteins, immunochemical analysis was utilized using antibodies against anti-spectrin, anti-beta-fodrin (non-erythroid spectrin), anti-protein 4.1 and anti-ankyrin antibodies. Immunoblot analysis of human epidermis with anti-spectrin and anti-beta-fodrin antibodies revealed that human epidermis contains 240 kDa and 235 kDa spectrin-like proteins, which might be identical to brain fodrin. Human epidermis also contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies, and contains ankyrin-like proteins of 210 kDa that cross react with anti-ankyrin antibodies. Analysis with immunofluorescence microscopy revealed that these antibodies reacted along the plasma membranes of human epidermal keratinocytes, eccrine sweat gland cells and sweat ductal cells. These results suggest that a membrane skeletal protein lattice might exist in these cells. Cultured human epidermal keratinocyte in the low Ca2+ medium (0.15 mM) showed that immunoreactive form of protein 4.1 and actin were present diffusely in the cytoplasm. When the cells were cultured with standardized Ca2+ medium (1.85 mM), protein 4.1 and actin were observed linearly along the cell margin and in the cytoplasm. Similar patterns of distribution were observed when anti-beta-fodrin antibody was used. Movement of membrane skeletal proteins from cytosol to the membrane suggest that these proteins or membrane skeletal lattice might play an important role in the formation of intercellular junctions.

Calcium↗
PubMed1996

High density sickle cell erythrocyte core membrane skeletons demonstrate slow temperature dependent dissociation.

We have previously demonstrated that slow dissociation of HDSS membrane skeletons in high ionic strength Triton X-100 buffer was related to a posttranslational modification in beta-actin, in which a disulfide bridge was formed between cysteine 284 and cysteine 373[Shartava et al: J Cell Bio 128:805, 1995]. These previous dissociation assays were limited to two homozygous (SS) sickle cell patients and a single temperature (37 degrees C). In the current work, we have expanded the SS subjects to 9 and have carried out dissociation assays at 0, 24, 30, 34, and 37 degrees C. At 0 degrees C there was limited dissociation of spectrin and actin from normal(AA), low density sickle cell(LDSS), and high density sickle cell (HDSS) core skeleton up to 24 hr. The first order rate constants for dissociation of spectrin, at 0 degrees C, was 0.030-0.035 x 10-4 sec-1 for AA,LDSS, and HDSS core skeletons. However at 24, 30, 34, and 37 degrees C the rate of dissociation of spectrin from HDSS core skeletons was significantly slower than the rate of dissociation from AA core skeletons. Having determined the first order rate constants for spectrin dissociation at these specified temperatures, we then asked whether dithiothreitol (DTT) would hasten the dissociation of core skeletons. The presence of DTT caused the rate of dissociation of the HDSS membrane skeleton to become statistically indistinguishable from the rate of dissociation of AA membrane skeletons. This is consistent with the suggestion that reversible thiol oxidation is responsible for the slow dissociation of the HDSS membrane skeleton.

Actins↗
PubMed2000

Integrin-type signaling has a distinct influence on NMDA-induced cytoskeletal disassembly.

Adhesion responses triggered by integrin-class matrix receptors have been implicated in the synaptic reorganization events necessary for certain types of neuronal plasticity. Hippocampal slice cultures were used to test whether the related structural transformations elicited by NMDA receptor stimulation are regulated by integrin-type signals. Infusing the slices with NMDA for a short period induced the expected disassembly of the cytoskeletal network, measured with antibodies that selectively recognize spectrin cleavage sites targeted by the protease calpain. Marked levels of the 150-kDa breakdown product (BDP) were produced, whereas concentrations of the parent spectrin were not changed. Interestingly, the calpain cleavage events were attenuated by 60% when integrin-type signaling was disrupted with the antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). This effect was RGDS-dependent, was largely evident in synapse-dense dendritic areas, particularly in subfield CA1, and was abolished when the NMDA exposure period was >5 min. These findings suggest that only those cytoskeletal alterations associated with brief synaptic activity are regulated by intact contact zones. AMPA-type glutamate receptors also were tested because, like spectrin, they are targets for calpain. Brief NMDA treatment caused a 15% loss of AMPA receptor GluR1 carboxytermini and this modification was augmented to 32% in the presence of GRGDSP. Thus, although blockage of matrix recognition signals decreased spectrin's susceptibility to disassembly, it increased the susceptibility of AMPA receptors to proteolysis. These data indicate that integrin-type signaling complexes are appropriately positioned to govern cytoskeletal reconfiguration while stabilizing the structural nature of AMPA receptors.

Animals↗
PubMed1978

Actin--membrane interactions: association of G-actin with the red cell membrane.

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.

Actins↗
PubMed1980

Expression of red cell membrane proteins in erythroid precursor cells.

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50-60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70-80% of all cells in the spleen of anemic animals, while only 1-2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.

Adult↗
PubMed2000

Erythroid phosphatidyl serine exposure is not predictive of thrombotic risk in mice with hemolytic anemia.

Thrombosis is a major complication of human hemolytic anemias such as sickle cell disease, thalassemia, and severe hereditary spherocytosis (HS). Mice with severe HS and severe hereditary elliptocytosis (HE) also suffer from thrombosis, with incidences ranging from 15 and 22% in beta-spectrin- and ankyrin-deficient mice, respectively, to 85 to 100% in alpha-spectrin-deficient and band 3 knockout mice. A contributing factor to thrombosis could be loss of phospholipid asymmetry of the mutant red blood cells (RBCs), with concomitant exposure of the aminophospholipid phosphatidylserine (PS). Increased PS exposure occurs in RBCs from sickle cell and thalassemia patients and in RBCs from band 3-deficient mice. To determine if increased PS exposure correlates with thrombotic risk in HS and HE mice with ankyrin, beta-spectrin, and alpha-spectrin deficiencies, measurements of FITC-labeled annexin V binding to externalized PS on RBCs were performed. PS exposure is elevated in all mice with HS and HE, but the percentage of RBCs with exposed PS does not correlate with thrombotic risk in these mice.

Age Factors↗
PubMed1994

Mechanism of control of erythrocyte shape: a possible relationship to band 3.

A mechanism of control of erythrocyte shape, differing significantly from those previously proposed, is hypothesized. It was inferred from previous observations on the states of association of Band 3, the anion exchange protein. Its salient points are as follows. (i) The membrane skeleton is a pliable protein meshwork structure used to generate different erythrocyte shapes. (ii) Band 3 is a dodecamer bound by one of its subunits to ankyrin which is itself bound to the mid region of filamentous spectrin. (iii) Spectrin, by virtue of its high flexibility, folds and unfolds and its folding links the two actin protofilaments bound at its ends to Band 3 through the intermediary of Band 4.1 and glycophorin A. (iv) The alternate influx and efflux of anions mediated by Band 3 respectively folds and unfolds spectrin. (v) The ratio of Band 3 molecules with outward-facing conformation to those with inward-facing conformation controls the extents of folding of the skeleton spectrin molecules. This ratio is governed by the Gibbs-Donnan equilibrium ratio of anions and protons and by the Band 3 anion affinities and exchange rates.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Cytoskeletal polarity in mammalian lymphocytes in situ.

The distribution of vimentin and spectrin in lymphocytes within murine lymphoid tissues was studied by means of immunofluorescence. A polarized submembranous aggregate of intermediate filaments was observed to be characteristic of lymphocytes within the medulla of the thymus as well as in lymphocytes within specific areas of spleen and lymph-node. This aggregate was determined to be in close association with a similarly polarized aggregate of spectrin. Lymphocytes of both B and T surface phenotype comprise the population of cells that are naturally polarized in terms of these cytoskeletal proteins. Lymphocytes with such a naturally polarized cytoskeleton are not observed in the spleen until approximately 5 days after birth, but are observed in the thymus by day 19 of gestation. Incubating lymphocytes with cytochalasin D, but not colchicine, caused a rapid dispersal of the spectrin aggregate without altering the polar accumulation of intermediate filaments. When splenic B-cells were allowed to form uropods as a result of ligand binding, the uropod (as well as surface receptor "cap") was positioned above the region containing the polar aggregate of spectrin and vimentin. The possible physiological significance of naturally occurring cytoskeletal polarity in lymphocytes is discussed.

Animals↗
PubMed1979

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates.

The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examing competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5 mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s). The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP. Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.

Caseins↗