Inhibition of mouse liver lipid peroxidation by high molecular weight phlorotannins from Sargassum kjellmanianum

The inhibitory effects of high molecularweight phlorotannins (HMP) from Sargassum kjellmanianum on mouse liverlipid peroxidation were investigated byspectrophotometric methods. The content ofmalondialdehyde (MDA) in liver samples wasmeasured by TBA (thiobarbituric acid)assay. It showed that HMP significantlyinhibited the generation of MDA invivo and in situations induced byCCl₄ and Fe²⁺-Vc (ascorbic acid),and significantly decreased membraneswelling of mouse liver mitochondria,compared with controls (p<0.01). HMP werefound to have strong anti-oxidativeactivity in inhibiting mouse liver lipidperoxidation.     

High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.     

High molecular weight microtubule-associated proteins within testes of hemipteran insects.

Results presented here indicate that the high molecular weight microtubule-associated proteins isolated from the ovaries of the hemipterans, Oncopeltus fasciatus and Notonecta glauca, while absent from nervous tissue, are present in the testes of the respective species. Here they are seen to be located to extensive microtubule aggregates within the cells surrounding the sperm, but not obviously to the sperm themselves.     

High molecular weight MAPs are part of the mitotic spindle

We have found that the microtubule-associated proteins of high molecular weight are located in the mitotic spindle. Indirect immunofluorescence studies reveal that the pattern of distribution of these proteins is similar to that described for tubulin and corresponds to the known phases of mitosis.     

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.     

Discrete soluble forms of middle and high molecular weight neurofilament proteins in dilute aqueous buffers

Neurofilament proteins purified from bovine spinal cord were characterized by sedimentation studies in aqueous buffers. In 10 mM Tris, pH 8, the middle molecular weight neurofilament protein (NF-M) has a sedimentation coefficient, S20,w, of 2.6 S. Sedimentation equilibrium data shows considerable nonideality; extrapolation to infinite dilution and correction for the primary charge effect yield a molecular weight of 1.09 X 10(5), indicative of a monomeric structure. When the ionic strength was increased, the sedimentation coefficient increased slightly, and the protein began to form larger aggregates. Reconstitution of short intermediate filaments was observed upon dialysis of denatured NF-M versus a reconstitution buffer. A circular dichroism spectrum of NF-M in 10 mM Tris was typical of alpha + beta proteins. High molecular weight neurofilament protein (NF-H) showed a considerable tendency to aggregate in 10 mM Tris, but a principal species with a sedimentation coefficient of 3.2 S was observed, and sedimentation equilibrium data also suggest a monomeric structure.     

Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal

The effects of the lipid peroxidation product 4-hydroxynonenal on freshly prepared human low-density lipoprotein (LDL) were studied. At a fixed LDL concentration (5.7 mg/ml) the amount of 4-hydroxynonenal incorporated into the LDL increased with increasing aldehyde concentration from 28-30 (0.2 mM) to 140 (1 mM) mol per mol LDL, whereas at a fixed aldehyde concentration (0.2 mM) its incorporation into LDL decreased with increasing LDL concentration from 48 (1 mg LDL/ml) to 26 (12 mg LDL/ml) mol 4-hydroxynonenal bound per mol LDL. Of the total hydroxynonenal taken up 78% was bound to the protein and 21% to the lipid moiety; the remaining 1% was dissolved as free aldehyde in the lipid fraction. Amino acid analysis of the apolipoprotein B revealed that 4-hydroxynonenal attacks mainly the lysine and tyrosine residues and to a lesser extent also serine, histidine and cysteine. Treatment of LDL with 4-hydroxynonenal results in a concentration-dependent increase of the negative charge of the LDL particle as evidenced by its increased electrophoretic mobility. Moreover, 4-hydroxynonenal treatment leads to a partial conversion of the apolipoprotein B-100 into higher molecular weight forms most probably apolipoproteins B-126 and B-151. Compared to malonaldehyde, 4-hydroxynonenal exhibits a much higher capacity to modify LDL and it is therefore believed that this aldehyde is a more likely candidate for being responsible for LDL modification under in vivo lipid peroxidation conditions.     

Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments

The mammalian neurofilament triplet proteins (210, 160 and 68 × 10³M_r proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 10³M_r protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 10³M_r protein to 68 × 10₃M_r protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 10₃M_r protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

High molecular weight forms of GTP-binding regulatory proteins from the bovine cerebellum

A new form of a low Km GTPase belonging to the family of regulatory GTP-binding G-proteins has been identified in bovine cerebellum. The molecular weight of this G-protein is several times as high as that of other G-proteins known to be alpha beta gamma heterotrimers: i. e., Gs, Gi, Go, transducin and a new G-protein which had recently been isolated in our laboratory from bovine cerebellum. The high molecular weight G-protein is stable against dissociation; its molecular mass does not change after treatment with DTT, colchicine and NaF. Using antibodies against the alpha-subunit of the formerly isolated cerebellar G-protein and the transducin beta-subunit, it was demonstrated that the both immunoreactive subunits are present in the high molecular weight G-protein. The two forms of the cerebellar G-proteins, i. e., "high" and "low molecular weight" ones, differ drastically in terms of the Mg2+ effect on their GTPase activity. Whereas at submicromolar concentrations of Mg2+ the GTPase activity of the former is virtually absent, the GTPase activity of the latter is more elevated in the presence of EDTA than in the presence of Mg2+.     

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestine.

The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE1) is rapidly metabolized in enterocytes. The degradation of HNE and other aldehydic products of lipid peroxidation processes seems to be an antioxidative defense system. The metabolism of HNE was studied in suspensions of rat enterocytes at 37 degrees C, pH 7.4 and at initial HNE concentration of 100 microM. About 70% of the HNE were degraded within three minutes of incubation. Main products of HNE which were identified in enterocytes were the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the 1,4-dihydroxynonene. Furthermore, the formation of metabolites of the tricarboxylic acid cycle is suggested. The quantitative share of HNE binding to proteins was low with about 1% of total HNE consumption after three minutes of incubation.     

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.     

Modification and inactivation of Cu,Zn-superoxide dismutase by the lipid peroxidation product,acrolein

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]     

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control相似文献   

The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.