Inactivation of Enzymes by Linoleic Acid Hydroperoxides and Linoleic Acid

The inactivation of the enzymes by linoleic acid hydroperoxides (LAHPO) was tested in connection with the toxicity of oxidized fat. At the same time, the inhibition of enzyme activities by linoleic acid was also tested. Ribonuclease (RNase), trypsin, chymotrypsin and pepsin which are considered to be simple proteins and not to be SH-enzymes were chosen as the enzymes. RNase was largely inhibited by LAHPO, but the other enzymes were inhibited by linoleic acid as well as LAHPO. The inhibition of each enzyme occurred at different pH. This fact may show that the inhibition occurs by binding of such hydrophobic compounds to the enzyme, and that the surface exposition of hydrophobic region may depend on the pH. Not only the reaction of some specific amino acid residue in the protein molecules with LAHPO, but also the binding of these hydrophobic compounds must be remembered in the mechanism of inhibition.     

The peroxidase nature of cytochrome P-420 utilizing a lipid peroxide substrate

The peroxidase properties of cytochrome P-420, in its “high spin” and “low spin” forms, have been investigated using N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as hydrogen donor, linoleic acid hydroperoxide (LAHPO) as substrate, and a preparation of cytochrome P-420 from hepatic microsomal “P-450 particles” as catalyst. The peroxidase reaction was found to be first order with respect to P-420 concentration and first order with respect to LAHPO concentration. Of the various hydroperoxides tested, only LAHPO was found to serve as a substrate for P-420.     

Measurement of Binding of Ascorbic Acid to Myrosinase by Rate of Dialysis

The antioxidative activities of l-amino acids on the methylene blue sensitized photooxidation of linoleic acid (LA) were studied in a 40% aqueous ethanol solution at pH 7.0 and 33°C. His and CysH inhibited the photosensitized oxidation of LA, and the antioxidative activity of His was larger than that of CysH. The ratio of the chemical reaction rate constant for His and singlet oxygen (¹O₂) to that for LA and ¹O₂ was similar to the ratio of the ¹O₂ quenching rate constant for His to that for LA. This fact shows that His inhibits the photosensitized oxidation of LA by quenching ¹0₂ chemically.

On the other hand, CysH reacted with linoleic acid hydroperoxides (LAHPO) or with and caused the decomposition of LAHPO or the formation of Cys. This fact suggests that CysH inhibits the photosensitized oxidation of LA by decomposing LAHPO or by quenching ¹O₂ chemically.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Effect of Linoleic Acid Hydroperoxides on Pepsin Activity

Pepsin was activated by incubating with linoleic acid hydroperoxides (LAHPO) at acidic region except at pH 4.0. Especially, the activation was best in pH 5.0 and 6.0 buffers, and the maximum activity attained was about 2 times of the original. It is still not certain whether LAHPO combined to the protein molecule or hydroperoxide groups reacted with some special amino acid residues in the protein molecule. The reactions were very sensitive to pH change and some conformational change of the protein molecule may be involved.     

Synthesis of the mannosyl-O-serine (threonine) linkage of glycoproteins from polyisoprenylphosphate mannose in yeast (Hansenula holstii)

The mannolipid synthesized from GDP-mannose and lipid acceptors in a particulate enzyme preparation from the yeast Hansenula holstii (R. K. Bretthauer, S. Wu, and W. E. Irwin, (1973) Biochim. Biophys. Acta, 304, 736–747) has the properties of dolicholmonophosphate mannose. Transfer of [¹⁴C]mannose from exogenously supplied, purified mannolipid to endogenous protein acceptors of the particulate enzyme fraction has now been demonstrated. The synthesis of radioactive products which are insoluble in chloroform-methanol and water is dependent upon time and concentrations of substrate, particulate fraction protein, and detergent. Addition of MgCl₂ or MnCl₂ to incubation mixtures prepared in the absence of these ions had only small stimulatory effects (20–25%), suggesting that the reaction is not dependent upon metal ions. Relatively high concentrations (0.005 m-0.05 m) of EDTA did partially inhibit the reaction, but this is considered to be due to secondary effects.Seventy percent of the radioactivity in the chloroform-methanol insoluble residue was solubilized with hot, neutral citrate buffer. The Chromatographic properties of this material on Sephadex gels and on DEAE-Sephadex were very similar to the properties of glycoprotein products derived from GDP-[¹⁴C]mannose. The chloroform-methanol insoluble products were also solubilized with Pronase which subsequently resulted in the isolation of a radioactive glycopeptide that contained 25% of the radioactivity transferred from mannolipid. The radioactive component of this glycopeptide was shown by β-elmination experiments and by amino acid analyses to be [¹⁴C]mannose residues linked O-glycosidically to serine and threonine residues. It was concluded, therefore, that one function of the mannolipid is to serve as mannosyl donor in the synthesis of the mannosyl-O-serine (threonine) linkage region of glycoproteins which may be part of the cell wall mannan-protein complex. Other mannose-containing products may also be synthesized from the mannolipid, as β-elimination of the chloroform-methanol insoluble fraction or of the Pronase soluble fraction did not result in recovery of all of the radioactivity as [¹⁴C]mannose.     

Synthesis of soluble, thylakoid, and envelope membrane proteins by spinach chloroplasts purified from gradients.

Intact spinach chloroplasts that had been purified on gradients of silica sol incorporated [³⁵S]methionine into soluble and membrane-bound products, using light as the sole energy source. The labeled chloroplasts were lysed osmotically and fractionated on a discontinuous gradient of sucrose into the soluble fraction and the thylakoid and envelope membranes. About 29% of the radioactivity in the chloroplast was recovered in the soluble fraction, 59% in the thylakoid membranes, and 0.1% in the envelope membranes. The products of protein synthesis in the different fractions, as well as in the whole chloroplast, were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. There were two zones of radioactivity in the gels of the soluble fraction, the major zone coincident with the large subunit of ribulose diphosphate carboxylase at a molecular weight of about 50,000. The thylakoid membranes contained five labeled polypeptides, the most active having a molecular weight of about 31,000. The envelope membranes contained a major radioactive component of a molecular weight of about 50,000 and two other minor components.     

Comparison of the Effects of Orally Administered Linoleic Acid,and Its Hydroperoxides and Secondary Autoxidation Products on Hepatic Lipid Metabolism in Rats

Linoleic acid, and its hydroperoxides and secondary autoxidation products were orally administered to rats (400 mg/rat). Their effects on hepatic lipid metabolism were examined. Linoleic acid reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase. It decreased the CoASH level and caused the accumulation of long-chain acyl-CoA. Hydroperoxides changed the compositions of unsaturated fatty acids in the hepatic lipids and lowered the content of neutral lipids. Secondary products stimulated carnitine palmitoyltransferase and decreased the content of neutral lipids. They reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase, and the levels of CoASH and acetyl-CoA. Thus, the effect of secondary products was apparently different from those of linoleic acid and its hydroperoxides.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

A study of ferritin iron formation in the rabbit alveolar macrophage

In order to investigate the intracellular pathway of iron to ferritin, rabbit alveolar macrophages were incubated with ⁵⁹FCl₃, homogenized by sonification, and a soluble cell fraction separated from the stroma by centrifugation at 23 000 g. The soluble fraction was examined by gel filtration using Sephadex. Two peaks were identified in the eluate at 254 nm; peak I contained a group of proteins, including ferritin, and most of the eluted radioactivity. The ⁵⁹Fe in this peak was confined to ferritin; no other ⁵⁹Fe-binding protein was identified. Peak II contained a small amount of ⁵⁹Fe. Chase experiments with ‘cold’ iron showed that peak I ⁵⁹Fe was derived from ⁵⁹Fe associated with the cell stroma. A protein carrier for ⁵⁹Fe between the stroma and ferritin was not identified in the eluate of the soluble fraction. Rather it appeared that iron moved from the stroma through the cytoplasm to ferritin in a low molecular weight form.     

Increased urinary excretion of 1-methyl-2-pyridone-5-carboxamide in rats administered 2-acetylaminofluorene.

The onset of fat accumulation within CCl₄ poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated.The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [³H]colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes or LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active.The presence of thiols, added to the incubation medium, partially protects against the inhibition of [³H]colchicine binding by aldehydes. This suggests that aldehydes act by reacting with -SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CCl₄ poisoning is discussed.     

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determins, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.     

A specific radioimmunoassay for vitellogenesis in mosquitoes

Hagedorn's assay for vitellogenesis, in which a crude antigen-antibody complex is collected directly on a Millipore membrane, without prior separation of soluble from insoluble proteins, is unspecific. This was proven as follows.Fat bodies of blood-fed mosquitoes (Aedes aegypti) were incubated in a medium containing ³H valine. The medium was analyzed for synthesis of vitellogenin by incubation with serum of normal rabbits (control) or of rabbits which were immunized against mosquito egg protein (antibody).When these mixtures were filtered directly through Millipore membranes (Hagedorn's method), the radioactivity in the control was about two-thirds of that in the antibody membrane. However, when the antigen-serum and antigen-antibody complex were centrifuged, the antigen-serum precipitate (control) contained only a very small percentage of the radioactivity present in the antigen-antibody precipitate. Apparently, the medium contained a large amount of soluble, nonvitellogenic protein that binds to the membrane.Analysis of the fat body homogenate showed that a large amount of the newly synthesized vitellogenin was stored intracellularly.Since all published data on vitellogenesis in mosquitoes depend on Hagedorn's assay, the validity of current concepts of the control of vitellogenesis in mosquitoes is open to question.     

PROTEIN SYNTHESIS IN VITRO IN NUCLEI OF HUMAN NERVOUS TISSUE

Abstract—

• 1 In vitro incorporation of tritiated leucine into nuclear proteins of normal human brain, astrocytoma I and II and glioblastoma multiforme was investigated. The distribution of radioactivity among various protein fractions of nuclei was determined.
• 2 The results demonstrate that the isolated nuclei of astrocytoma I and II incorporate radioactivity into proteins at least 40 times more actively than nuclei of normal human brain or glioblastoma multiforme.
• 3 The residual protein fraction was the most highly labelled fraction among the nuclear pioteins. This fraction from astrocytomas incorporates relatively more radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The buffered-saline soluble protein fraction of astrocytoma nuclei contained a relatively lower amount of radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The total radioactivity incorporated into the deoxyribonucleoproteins seemed to increase with the malignancy of the tissue investigated. The significance of the results with respect to malignant transformation is discussed.

     

Dégradation d'acides aminés à l'interface eau-sédiment réconstitué d'une lagune méditerranéenne (Golfe du Lion)

Degradation of Amino Acids at a Simulated Water-sediment Interface of a Mediterranean Lagoon Environment, Golfe du Lion The degradation of two ¹⁴C and ³H labelled amino acids at the simulated water-sediment interface of a Mediterranean lagoon was studied. The four day experiments included the respiratory activity measurement and the study of incorporation processes of the radioactivity in specific organic fractions of the ¹⁴C arginine. After active mineralization occurred during the first day, the radioactivity in the acid-soluble fraction decreased and polycondensed products were progressively incorporated. The radioactivity of the ³H lysine was included mainly in the acid soluble fraction. The nature of the substrate, and the reducing conditions in the environment affect the radioactivity distribution in the organic fractions. The identification of amino acids in the acid-soluble and base soluble fractions shows that the labelled arginine and lysine and other amino acids in the acid soluble and base soluble fractions shows that the labelled arginine and lysine released from the initial compounds are important quantitatively.     

On the mechanism of oxidation of cholesterol at C-7 in a lipoxygenase system.

Incubation of [7-2H2]cholesterol with soybean lipoxygenase and linoleic acid in the presence of oxygen gave a mixture of 5-cholestene-3 beta,7 alpha-diol, 5-cholestene-3 beta,7 beta-diol, 3 beta-hydroxy-5-cholesten-7-one,5 alpha,6 alpha-epoxycholestan-3 beta-ol, and 5 beta,6 beta-epoxycholestan-3 beta-ol. The conversion into the 7-oxygenated products was associated with a very high intermolecular isotope effect (KH/KD = 15-17), suggesting that the rate-limiting step in the overall conversion is likely to be the abstraction of hydrogen at C-7 in a radical reaction. Evidence that linoleic acid is to some extent directly involved was obtained with the use of [7-3H]cholesterol. Incubation of [7-3H]cholesterol resulted in a significant incorporation of 3H in the reisolated linoleic acid fraction. The isotope effect associated with conversion of [7 alpha-2H]cholesterol into 7-oxygenated products in the lipoxygenase system was 2-3, indicating that the extraction of hydrogen is nonstereospecific. Incubation of [7-2H2]cholesterol with 13-hydroperoxy-9,11-octadecadienoic acid gave the above 7-oxygenated products with relatively small isotope effects (KH/KD = 3-4). It is concluded that the most important mechanism for oxidation of cholesterol at C-7 in the lipoxygenase system involves participation of radicals and that a carbon-centered linoleic acid radical can extract hydrogen directly from cholesterol. Fatty acid hydroperoxides and their secondary products seem to be less important as initiators in connection with oxidation of cholesterol.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Incorporation of 3H-Gibberellin A20 in roots of G2 peas

Following application of ³H-Gibberellin A₂₀ (GA₂₀) to roots of G2 pea seedlings and homogenization of the roots, about 3% of the radioactivity in the tissue could be precipitated from a 30,000 × g supernatant with trichloroacetic acid (TCA) (soluble fraction) while about 5% of the radioactivity pelleted at 30,000 × g (particulate fraction). The radioactivity in the particulate fraction was soluble in sodium dodecyl sulfate (SDS), but was not dialyzable and was insoluble in ethanol. Electrophoresis of the soluble fraction gave only one band of radioactivity, while that of the particulate fraction gave multiple bands. Acid hydrolysis of the soluble fraction released radioactivity that ran coincident with acid-treated GA₂₀ on silicic-acid column chromatography. The particulate fraction gave numerous radioactive peaks following acid hydrolysis, two of which were coincident with GA₂₀ and GA₂₉ (hydroxylation product of GA₂₀) on silicic acid chromatography. Treatment of the particulate and soluble fractions with RNase, DNase, and proteases showed a significant solubilization of radioactivity only with the proteases, suggesting that the GA is bound to a proteinaceous macromolecule. Complete proteolytic hydrolyis followed by thin layer chromatography showed 65% of the radioactivity from the soluble fraction running separately from free GAs or the individual amino acids; the particulate fraction gave mainly (60%) free GAs on enzymatic hydrolysis and much smaller amounts (17%) in a position separate from that of the GAs or amino acids. Binding of ³H-GA to protease-sensitive material was obtained with biologically active ³H-GA₂₀ and ³H-GA₁.