Conventional antibody against Nepsilon-(carboxymethyl)lysine (CML) shows cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL): immunochemical quantification of CML with a specific antibody

Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.     

Definition of the immunogenic forms of modified human LDL recognized by human autoantibodies and by rabbit hyperimmune antibodies

Humans and laboratory animals recognize human modified LDL as immunogenic. Immune complexes (ICs) isolated from human sera contain malondialdehyde-modified LDL (MDA-LDL) and N (epsilon)(carboxymethyl)lysine-modified LDL (CML-LDL) as well as antibodies reacting with MDA-LDL, copper-oxidized LDL (OxLDL), CML-LDL, and advanced glycosylation end product (AGE)-modified LDL. OxLDL and AGE-LDL antibodies isolated from human sera recognize the same LDL modifications and do not react with modified non-LDL proteins. Rabbit antibodies have different reactivity patterns: MDA-LDL antibodies react strongly with MDA-LDL and MDA-BSA but weakly with OxLDL; OxLDL antibodies react strongly with OxLDL and weakly with MDA-LDL; CML-LDL antibodies react with CML-LDL > CML-BSA > AGE-LDL > OxLDL; AGE-LDL antibodies react strongly with AGE-LDL, react weakly with OxLDL, and do not react with CML-LDL. Thus, human and rabbit antibodies seem to recognize different epitopes. Capture assays carried out with all rabbit antibodies showed binding of apolipoprotein B-rich lipoproteins isolated from ICs, suggesting that laboratory-generated epitopes are expressed by in vivo-modified LDL, although they are not necessarily recognized by the human immune system. Thus, the definition of immunogenic forms of modified LDL eliciting human autoimmune responses requires the isolation and characterization of autoantibodies and modified LDL from human samples, whereas rabbit antibodies can be used to detect in vivo-modified human LDL.     

Aging increases Nepsilon-(carboxymethyl)lysine and caloric restriction decreases Nepsilon-(carboxyethyl)lysine and Nepsilon-(malondialdehyde)lysine in rat heart mitochondrial proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

Aging Increases N epsilon -(Carboxymethyl)lysine and Caloric Restriction Decreases N epsilon -(Carboxyethyl)lysine and N epsilon -(Malondialdehyde)lysine in Rat Heart Mitochondrial Proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

Identification of N epsilon-(carboxyethyl)lysine,one of the methylglyoxal-derived AGE structures,in glucose-modified protein: mechanism for protein modification by reactive aldehydes

We have developed a separation system for N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) by HPLC equipped with a styrene-divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N(alpha)-acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.     

Binding of [99mTc]chondroitin sulfate to scavenger receptors on human chondrocytes as compared to binding of oxidized [125I]LDL on human macrophages

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with 99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37 degrees C for 2 h revealed the presence of high-affinity binding sites for CS with a Kd of 2.3 x 10(-9) mol/L and a Bmax of 5.0 x 10(8). Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 +/- 19.5% and of the number of CS binding sites/cell by 62.0 +/- 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [125I]-LDL binding we found a Kd of 0.45 x 10(-8) mol/L and a Bmax of 0.14 x 10(6) on quiescent monocytes and for [125I]-(ox)LDL binding a Kd of 1.8 x 10(-8) mol/L and a Bmax of 1.3 x 10(6) using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein-proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).     

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of glycated proteins: age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of fructoselysine (FL) in glycated (nonenzymatically glycosylated) proteins in vitro and has also been detected in human tissues and urine [Ahmed et al. (1986) J. Biol. Chem. 261, 4889-4894]. In this study, we compare the amounts of CML and FL in normal human lens proteins, aged 0-79 years, using specific and sensitive assays based on selected ion monitoring gas chromatography-mass spectrometry. Our results indicate that the lens content of FL increases significantly between infancy and about age 5 but that there is only a slight, statistically insignificant increase in FL between age 5 and 80 (mean +/- SD = 1.4 +/- 0.4 mmol of FL/mol of Lys). In contrast, the lens content of the oxidation product, CML, increased linearly with age, ranging from trace levels at infancy up to 8 mmol of CML/mol of lysine at age 79. The ratio of CML to FL also increased linearly from 0.5 to 5 mol of CML/mol of FL between age 1 and 79, respectively. These results indicate that CML, rather than FL, is the major product of glycation detectable in adult human lens protein. The age-dependent accumulation of CML in lens protein indicates that products of both glycation and oxidation accumulate in the lens with age, while the constant rate of accumulation of CML in lens with age argues against an age-dependent decline in free radical defense mechanisms in this tissue.     

人红细胞生成素单克隆抗体的制备、鉴定及应用研究

用rhEPo作为抗原,免疫BALB／c小鼠,取其脾细胞与x63Ag8．653小鼠骨髓瘤细胞融合,再碱性PAGE方法进一步分离并纯化的rhEpo,包被Pvc板,对杂交瘤用ELlSA方法进行筛选,获得两株稳定分泌抗hEPO单抗的杂交瘤细胞株。经鉴定分别属于IgG1、IgG2b,轻链均为k链,Kd分别为5．53×10^-10mol／L和1．34×1O^-10mol／L．用western blot方法证明两者对hEPO具有高度韵专一性．能特异地识别rhEPO和尿源hEPO。所制备单抗可作为亲和层析的配体,用于再生障碍性贫血病人尿中EPO及哺乳类工程细胞所表达的hEPO的分离、纯化,并可用于hEPO的定量检测．     

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.     

Effect of isoflavone genistein on insulin receptors in perfused liver of ovariectomized rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein, insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd). BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610 +/- 77 x 10(-15) (no genistein) to 238 +/- 72 x 10(-15) mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372 +/- 59 x 10(-15) mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421 +/- 46 x 10(-15) mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM--1.44 +/- 0.18 x 10(-9) mol/l; perfusion with BMP + genistein--0.83 +/- 0.20 x 10(-9) mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.     

Chemoselective synthesis of peptides containing major advanced glycation end-products of lysine and arginine.

Useful methodologies have been developed, enabling the straightforward synthesis of peptides containing N(epsilon)-(carboxymethyl)-L-lysine (CML) and N(epsilon)-(carboxyethyl)-L-lysine (CEL), the major glycation end-products of lysine. These lysine derivatives were successfully incorporated into growing peptide chains via standard Fmoc/Ot-Bu peptide synthesis procedures. For the synthesis of peptides containing major glycation end-products of arginine, synthetic routes have been developed enabling the transformation of ornithine residues in peptides into the well-known arginine-derived advanced glycation end-products (AGEs) Glarg, carboxymethyl-L-arginine (CMA), MG-H1, MG-H2, MG-H3, and carboxyethyl-L-arginine (CEA), respectively, by means of special modifying agents. Furthermore, it was shown that Glarg-containing peptides become quantitatively hydrolyzed into CMA-peptides under physiologic conditions. A similar reaction was observed in case of a MG-H3-peptide, which turned into a CEA-peptide under these conditions.     

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.     

Identification and quantification of N(epsilon)-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry

The identification and quantification of N(epsilon)-(hexanoyl)lysine (N(epsilon)-HEL), which was found from the reactions between lipid hydroperoxide and lysine, from human urine was examined using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The N(epsilon)-HEL in the partially purified urine fraction was identified using LC/MS/MS by several approaches including precursor/product ion scans. The peak found by the multiple-reaction monitoring (MRM) of the collision-induced fragmentation of N(epsilon)-HEL was clearly observed in urine, and the elution position coincided with the synthetic standard N(epsilon)-HEL. The product, estimated N(epsilon)-HEL, was absorbed by a specific antibody to N(epsilon)-HEL. Moreover, N(alpha)-HEL, one of the plausible hexanoyl adducts from the reaction between the N(alpha) moiety of L-lysine and the peroxidized lipid, was hardly detected in urine samples, suggesting that the origin of the N(epsilon)-HEL is the peroxidized lipid-modified proteins but not artificial hexanoylated L-lysine. Using the MRM technique, the amount of urinary N(epsilon)-HEL from the control subjects (observed healthy) was estimated to be 1.58 +/- 0.23 mumol/mol of creatinine. A comparative study of the urinary N(epsilon)-HEL with an oxidative stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, showed a high correlation (r = 0.844) between the two biomarkers. Furthermore, the quantification of N(epsilon)-HEL in the control and diabetic urines revealed that the urinary N(epsilon)-HEL from diabetic subjects (3.21 +/- 0.65 mumol/mol of creatinine) was significantly higher than that from the control subjects.     

The immune response to GHRH, relationship to conformation

Antibodies to GHRH1-44, GHRH1-29, and proinsulin were induced in guinea pigs. GHRH1-44 forms 7 S and 10 S complexes with antibodies. It is a divalent antigen. The sequence 30-44 bound 85% of the antibodies to GHRH1-44 with high affinity (3.8 +/- 0.9 x 10(9) l/mol). The fragment 1-29 bound with low affinity (0.6 +/- 0.3 x 10(9) l/mol) 15% of the antibodies (2p less than 0.001). Antibodies to GHRH1-29 had low affinity towards the native hormone (0.4 +/- 0.2 x 10(9) l/mol) and the region 1-29 (0.3 +/- 0.2 x 10(9) l/mol). Antibodies to proinsulin bound linear C-peptide with lower affinity (0.3 +/- 0.2 x 10(9) l/mol) than the C-peptide loop in proinsulin (3.4 +/- 0.9 x 10(9) l/mol). It is concluded that the conformation of the epitopes on the sequence 1-29, recognized during the immune response, i.e. on the cell membrane, is different from the conformation of GHRH1-29 or GHRH1-44 in aqueous solution.     

alpha-lactalbumin mutant acting as lysozyme

A mutant of alpha-lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in alpha-lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP-(NAcGlc)(3), with a K(M) and k(cat) of 0.160 +/- 0.00986 mmol/L and 3.39 +/- 0. 0456 x10(-5) min(-1), respectively, which was comparable with those of chicken lysozyme of 0.137 +/- 0.0153 mmol/L and 5.25 +/- 0.115 x10(-4) min(-1). By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in alpha-lactalbumin could be converted into an enzyme that was 17.5-fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond.     

Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies.

The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.