Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes.

The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.     

Intra- and intermolecular electron transfer processes in redox proteins

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.     

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.     

Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by cross-linking of membrane proteins

The effects of cross-linking of membrane proteins on hemolysis of human erythrocytes under high pressure (2.0 kbar) were examined. The membrane proteins were cross-linked by oxidation of their SH-groups with diamide (0.05-0.5 mM) under different pressures (1-1,000 bar) at which no hemolysis occurs. As the pressure during diamide treatment was raised, the degree of hemolysis under 2.0 kbar and the quantity of cytoskeletal proteins extracted in a low ionic strength medium were gradually decreased. However, both values were increased by reduction with dithiothreitol. From the determination of membrane SH-groups, it was found that cross-linking of membrane proteins by diamide was accelerated under pressure. Only in erythrocytes treated with diamide under pressure were parts of spectrin and ankyrin, in addition to band 3 and band 4.2 proteins, extracted by using Triton X-100. One- and two-dimensional SDS-PAGE of membrane proteins showed that cross-linking of the membrane with cytoskeletal meshwork through linking proteins, in addition to that of membrane proteins themselves, was formed only in the diamide treatment under pressure. These results indicate that pressure-induced hemolysis is greatly suppressed by the supramolecular-weight polymers formed among membrane proteins, and that the high pressure technique is useful for cross-linking membrane proteins with diamide.     

Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins

Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS–PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.     

A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.     

The interaction of 1-fluoro-2,4-dinitrobenzene with amino-phospholipids in membranes of intact erythrocytes,modified erythrocytes,and erythrocytes ghosts

Summary 1-Fluoro-2,4-dinitrobenzene (FDNB) has been used to study the availability of amino-containing phospholipids in erythrocyte membranes and ghosts in an aqueous, isotonic medium. It was found that the addition of bovine serum albumin (BSA) to the medium protects the cells from cation leak and protects some of the amino-phospholipids from reacting with the probe. In isotonic medium without BSA, 46% of the phosphatidylethanolamine and 12% of the phosphatidylserine of erythrocytes and 73% and 21% of these respective lipids of ghosts react with the probe. In the presence of 70 m BSA, 31% of phosphatidylethanolamine and 6.5% of phosphatidylserine of erythrocytes and 59% and 16% of these respective lipids of ghosts react with the probe. The labeling of these lipids does not change under conditions of varying tonicity, or after treatment of erythrocytes with pronase or lysolecithin. The data suggest that 46% of phosphatidylethanolamine and 12% of phosphatidylserine of the erythrocyte membrane are free in a lipid bilayer; 27% and 9% of these respective lipids are loosely bound to proteins which are lost during the preparation of ghosts and 27% of the phosphatidylethanolamine and 79% of the phosphatidylserine are tightly bound to core proteins which remain part of the erythrocyte membrane even after hemolysis.     

Photodynamic effects of protoporphyrin on human erythrocytes. Nature of the cross-linking of membrane proteins

Protoporphyrin-sensitized photooxidation in human red blood cell membranes leads to severe deterioration of membrane structure and function. The membrane damage is caused by direct oxidation of amino acid residues, with subsequent cross-linking of membrane proteins. The chemical nature of these cross-links was studied in model systems, isolated spectrin and red cell ghosts. Cysteine and methionine are not involved in the cross-linking reaction. Further it could be shown that dityrosine formation, the crucial mechanism in oxidative cross-linking of proteins by peroxidase-H2O2 treatment, plays no role in photodynamic cross-linking. Experimental evidence indicated that a secondary reaction between free amino groups and a photooxidation product of histidine, tyrosine or tryptophan is involved in photodynamic cross-linking. This was deduced from the reaction observed between compounds containing a free amino group and photooxidation products of these amino acids, both in model systems, isolated spectrin and erythrocyte ghosts. In accordance, succinylation of free amino groups of membrane proteins or addition of compounds with free amino groups protected against cross-linking. Quantitative data and consideration of the reaction mechanisms of photodynamic oxidation of amino acids make it highly probable that an oxidation product of histidine rather than of tyrosine or tryptophan is involved in the cross-linking reaction, via a nucleophilic addition by free amino groups.     

Reassociation of membrane glycoprotein with intact erythrocytes

Purified membrane glycoprotein was found to specifically bind to erythrocyte surfaces. (1) The glycoprotein showed over 80-fold greater binding to erythrocytes than did bovine serum albumin; (2) the binding of the glycoprotein was not significantly inhibited by the presence of other proteins even when added in 175-fold greater concentration; (3) the glycoprotein's binding showed both time and concentration dependence. Radiolabeled glycoprotein was re-isolated after binding to erythrocytes and, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 64% of the label was recovered from the glycoprotein bands. The glycoprotein preparation would bind to erythrocyte surfaces to the extent of 6–7% of that naturally occurring in the membranes.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Probing actin polymerization by intermolecular cross-linking

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.     

Oxidative cross-linking of plasma membrane arabinogalactan proteins

Monoclonal antibodies which recognize carbohydrate in arabinogalactan proteins (AGPs) have revealed that certain carbohydrate epitopes at the outer plasma membrane surface are demonstratively regulated. Some epitomes are expressed according to cell position, and AGES are thought to play a role in cell—cell interaction during development. This study demonstrates that sugar beet plasma membranes contain two subagencies of AGES, with apparent molecular masses of 82 and 97 kDa, and that each subfamily consists of a small number of acidic AGP isoforms. Excision of leaves generates three additional AGP complexes with apparent molecular masses of 120, 170 and 210 kDa, with the 170 kDa complex being the major form induced by excision. The addition of millimolar concentrations of H₂O₂ to a partially purified fraction of the 82 and 97 kDa AGPs also generates AGP complexes, with the 170 kDa complex as the major form. These results indicate that the plasma membrane AGPs are a target for endogenous H₂O₂.     

Protein-protein contacts in solubilized membrane proteins, as detected by cross-linking

The amount of detergent required for the solubilization of membrane proteins needs to be optimised as an excess may cause loss of activity and insufficiency may result in poor solubilization or heterogeneous samples. With sarcoplasmic reticulum Ca2+ -ATPase as an example we show by cross-linking that it can be misleading to choose the proper amount of detergent based on clarification of membrane suspensions, because clarification -as detected by turbidity measurements, for instance- precedes full protein solubilization as monomers. We demonstrate that to assess the extent of sample homogeneity at a given detergent/protein ratio, cross-linking followed by HPLC gel filtration in detergent usefully complements cross-linking followed by SDS-PAGE.     

Transglutaminase-catalysed cross-linking of proteins phosphorylated in the intact glucose-stimulated pancreatic beta-cell

Incubation of intact islets in the presence of [32P]Pi and stimulatory levels of glucose followed by separation of phosphorylated islet proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of a high molecular weight phosphopolymer which did not transverse a 3% (w/v) acrylamide gel. The majority of this phosphopolymer (approx. 70%) was present in the 600 x g sedimented fraction of islet homogenates. Islet homogenates obtained from intact islets previously incubated with [32P]Pi and stimulatory levels of glucose when incubated under conditions that activated the islet transglutaminase resulted in an increase in the amount of phosphopolymer present in the 600 x g sedimented fraction. Inhibitors of transglutaminase activity which are known to inhibit glucose-stimulated insulin release led to a significant reduction in the fraction of phosphopolymer present in the glucose-stimulated intact islet. These findings suggest that protein cross-linking and phosphorylation reactions may be closely linked in the pancreatic beta-cell.