3-methylbutanal Research Articles

Biochemical activities of Micrococcaceae and their effects on the aromatic profiles and odours of a dry sausage model

Micrococcaceae are used as starter cultures in fermented meat, it is therefore important to determine their biochemical activities and especially their aroma producing potential. With this aim, 19 strains of Micrococcaceae were studied in a dry-sausage model. Odours of the samples were assessed by a trained panel. Volatile compounds were analysed by dynamic headspace gas chromatography–mass spectrometry. Strains belonging to the species Staphylococcus warnerior Staphyloccocus saprophyticusthat were both lipolytic and unable to reduce nitrate, developed putrid, nauseous or bread odours. These samples were characterized by high desorption of aldehydes (heptanal, hexanal, octanal), 3-hydroxy butan-2-one and 2, 3-butanedione. The highest dry-cured odour was developed with the inoculation of strains of Staphylococcus carnosusand Staphylococcus xylosuswhich had low lipolytic and proteolytic activities and did not produce acetoin but effectively reduced nitrate. Their inoculation in the sausage model was associated with the greatest desorbtion of 3-methyl butanal, methylketones and ethyl esters and the lowest desorption of alkanes and 3-hydroxy butan-2-one and 2, 3-butanedione.

Comparison of Postmortem Autolysis in Cardiac and Skeletal Muscle

To understand the mechanism in postmortem autolysis better, processes in the postmortem degradation of myofibril proteins in the presence of protease inhibitors were studied. Male Wistar rats were given injections of the carboxyl-, thiol-, and serine-protease inhibitors, pepstatin, Ep-475[L-transepoxysuccinyl-leucylamide(3-methyl) butane; E-64-C], and chymostatin, via the femoral vein. Control rats were similarly treated with saline. Then, myofibril proteins were isolated from their cardiac and femoral muscles and from those of control animals at various times after death, and degradation of these myofibril proteins with time was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In cardiac muscle, alpha-actinin was degraded rapidly, followed by the heavy chain of myosin and light chain of myosin (L2). Actin and the light chain of myosin (L1) were degraded slowly. the degradations of the heavy chain of myosin, alpha-actinin, tropomyosin and L2 after 14 days were not inhibited by pepstatin, but were inhibited by Ep-475 and chymostatin. In skeletal muscle, L1 and L2 were degraded rapidly, followed by the heavy chain of myosin and alpha-actinin. Actin was degraded slowly and was still unchanged 2 weeks after death. The degradations of protein components were inhibited by pepstatin, Ep-475 and chymostatin. These results indicated that after death the components of myofibrils are degraded by various proteases at various rates depending on their properties or structures. This degradation is fundamentally the same in cardiac and skeletal muscles, but inhibitors have somewhat different effects on the postmortem degradation processes after death in the two types of muscle.

Identification and Localization of a Novel Cathepsin S-like Proteinase in Guinea Pig Spermatozoa

Guinea pig spermatozoa were found to contain a novel cysteine proteinase that most closely resembled lysosomal cathepsin S. The responsible enzyme was shown to have an acrosomal localization. Like the cathepsin S of lymphocyte-rich tissues, e.g., lymph nodes and spleen, the spermatozoal enzyme digested protein substrates such as hemoglobin and Azocoll optimally at about pH 3.5 in the presence of pepstatin. No appreciable action was manifested in the range of pH 5-6 on protein substrates nor on the 7-(4-methyl)coumarylamide (NMec) derivatives of peptides commonly employed for the fluorometric assay of cathepsins B (benzyloxycarbonyl (Z)-Arg-Arg-NMec), H (Arg-NMec), and L (Z-Phe-Arg-NMec). Like spleen cathepsin S, the sperm enzyme selectively hydrolyzed Z-Phe-Arg-NMec, but its activity was uncharacteristically limited to a pH range of 3.0-3.5, where it displayed a typical high sensitivity to sulfhydryl reagents (HgCl 2, mersalyl acid, iodoacetate), leupeptin, Z-Phe-Phe-CHN 2, and L- trans-epoxysuccinylleucylamido(3-methyl)butane. Gossypol (a male antifertility agent) was also inhibitory. Inhibition by the mercurial sulfhydryl reagents was completely reversible with dithiothreitol. Pepstatin, a potent inhibitor of aspartic proteinases, and serine proteinase inhibitors (phenylmethylsulfonyl fluoride, beazamidine) were ineffective. Other properties displayed by the sperm extract that were uniquely characteristic of cathepsin S included stability under alkaline conditions, and a greater rate of hydrolysis when the P 2-phenylalanine of the assay substrate was replaced by two aliphatic residues, as in Z-Leu-Leu-Arg-NMec.

Iodo and diiodotyrosine epoxysuccinyl derivatives as selective inhibitors of cathepsin B

Eight new analogs of l- trans-epoxysuccinyl- l-leucylamido(3-methyl)butane (E-64-c) containing Phe, Tyr, Tyr(1) or Tyr(I 2) in place of Leu, were synthesized and tested as inhibitors of papain, bovine spleen cathepsin B, calpain I and II from porcine red cells and porcine kidney, respectively. By use of kinetic methods, the new E-64 analogs proved to irreversibly inactivate both papain and cathepsin B via reversible enzyme-inhibitor intermediates EI. Second-order rate constants for inactivation were in the range 3500-55 100 M −1s −1 for papain and 650–105 000 M −1s −1 for cathepsin B. For the inactivation of calpain I and II they ranged between 250 and 2000 M −1s −1 and were similar to those of the known E-64-c. The effectiveness of the amino acid contained in the inhibitors tested increased in the order Tyr(I) ≈ Tyr(I 2) < Tyr < Phe < Leu for papain and Phe < Tyr < Tyr(I) < Leu < Tyr(I 2) for cathepsin B inactivation. Replacement of the l with the d- trans-epoxysuccinyl unit caused a 10–100-fold decrease in inhibitor potencies.

The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.

Lysosomal cysteine endopeptidases mediate interleukin 1-stimulated cartilage proteoglycan degradation

The peptidyl diazomethane inactivator of cysteine endopeptidases, benzyloxycarbonyl-Tyr-Ala-CHN2, was tested as an inhibitor of interleukin 1 alpha-stimulated release of proteoglycan from bovine nasal septum cartilage explants. Like the previously tested epoxidyl peptide proinhibitor trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester, it proved to be an effective inhibitor of proteoglycan release from cartilage, with significant inhibition at a concentration of 1 microM. The inhibition did not seem to be due to a general toxic effect. The rates of inactivation of the bovine cysteine endopeptidases by the peptidyl diazomethane, the epoxidyl peptide proinhibitor and its active form were determined. Benzyloxycarbonyl-Tyr-Ala-CHN2 proved to be a rapid inactivator of cathepsins L, S and B, but reacted much more slowly with cathepsin H and calpain. Thus it would appear that the latter two enzymes are not implicated in proteoglycan release in our test system. The peptidyl diazomethane and epoxidyl peptide proinhibitor (above) were also tested for their effects on three other interleukin 1-mediated cellular events, namely epidermal growth factor receptor transmodulation, and interleukin 6 and prostaglandin E2 production. In all cases the inactivators did not interfere with the response to interleukin 1 in human gingival fibroblasts. We conclude that one or more of the lysosomal cysteine endopeptidases cathepsins B, L and S mediate interleukin 1-stimulated cartilage proteoglycan degradation without affecting signal transduction.

Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases

Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine-stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester (Ep453), a 'prodrug' of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes.

Effects of Tuber metabolites on the rhizospheric environment

Several species of Tuber (Ascomycotina, Tuberales) produce a ‘burned’ area around their symbiotic plants as a result of a phytotoxic action. The volatile substances from Tuber sporophores, truffles, have been previously identified by gas chromatography coupled to gas mass spectrometry and then tested for their toxic effects on higher plants as well as soil micro-organisms. All tested organisms appeared to suffer a great inhibition when exposed to some of these substances evaporated into desiccators. Three aldehydes (2-methyl propanal, 2-methyl butanal and 3-methyl butanal) produced these effects on the two soil fungi tested ( Penicillium vinaceum and Aspergillus alliaceus ). In addition to some of these, two alcohols (2-methyl butanol and 3-methyl butanol) are also active on plants ( Triticum vulgare and Lens culinaris ). 2-Methyl butanol generally affects plants to a high degree, but the strongest effects to microfungi are caused by 3-methyl butanal. However, a strain of Pseudomonas isolated from truffle surface tolerated much higher concentrations of the three aldehydes. The substances affecting the soil micro-aerobic environment, where seeds germinate and roots live, are normal compounds of fungal metabolism produced via pyruvate pathway. Their toxic action could be a consequence of alterations caused both to the cellular membrane and to nucleic acids. The new term ‘hydnosphere’ has been proposed to define this peculiar case of soil relationships between truffle and neighbouring coenosis.

Evidence suggesting a role for cathepsin L in an experimental model of glomerulonephritis

We have utilized specific, irreversible inhibitors of cysteine proteinases to examine the role of renal cathepsin B and cathepsin L in the proteinuria which occurs in an experimental model of human glomerular disease. Administration of trans-epoxysuccinyl- l-leucylamido-(3-methyl)butane (Ep475) a specific, irreversible inhibitor of cysteine proteinases, including cathepsins B and L, significantly reduced proteinuria in rats with experimentally induced, neutrophil-independent, anti-GBM antibody disease (controls: 10 ± 1 mg/24 h, N = 8; anti-GBM antibody disease: 203 ± 30 mg/24 h, N = 8; anti-GBM antibody disease + Ep475:112 ± 13 mg/24 h, mean ± SEM, N = 6, P < 0.05). There was a marked reduction in the activity of both cathepsin B and cathepsin L in renal cortices obtained from Ep475-treated rats compared to either saline-treated controls or rats treated with anti-GBM IgG only. Administration of Z-Phe-Tyr( O-t-butyl)CHN 2, a specific, irreversible cysteine proteinase inhibitor with a high degree of selectivity toward cathepsin L, also caused a reduction in anti-GBM antibody-induced proteinuria (90 ± 18 mg/24 h, N = 6, P < 0.05). This reduction in proteinuria was accompanied by a marked decrease (−84%) in the specific activity of renal cortical cathepsin L in Z-Phe-Tyr( O-t-butyl)CHN 2-treated rats. However, cathepsin B activity was unchanged. There was no significant change in the renal anti-GBM antibody uptake, plasma urea nitrogen, or plasma creatinine values in the Z-Phe-Tyr( O-t-butyl)CHN 2 -treated rats compared to rats treated with anti-GBM IgG only or saline-treated controls. These data document the ability of cysteine proteinase inhibitors to decrease the proteinuria which occurs in a neutrophil-in-dependent model of human anti-GBM antibody disease and suggest an important role for cathepsin L in the pathophysiology of the proteinuria which occurs in this model.

Cathepsin L — A latent proteinase in guinea pig sperm

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.

Mechanism of postmortem autolysis of skeletal muscle

Male Wistar rats were treated with the carboxyl, thiol, and serine protease inhibitors, pepstatin, Ep-475[ l-trans-epoxysuccinyl-leucylamide(3-methyl) butane; E-64-c], and chymostatin. Then the femoral muscles of these rats and control animals were used for preparation of myofibril proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the degradation of these myofibril proteins with time (day) after death. The protease activities of the muscle were also measured. Tropomyosin was degraded most rapidly, followed by the heavy chain of myosin, α-actinin, and light chains of myosin (L1 and L2). Actin and troponin-T were degraded slowly, still remaining unchanged 2 weeks after death. The degradation of protein was not inhibited by pepstatin but was inhibited strongly by Ep-475 and very strongly by chymostatin. Chymostatin inhibited degradation of all components except α-actinin more strongly than Ep-475. Data on enzyme activities were consistent with these findings. These results suggest that after death the components of myofibrils are degraded with various proteases at various rates depending on their properties or their structure and that the proteases involved in the degradation show some specificity.

PRODUCTION OF FLAVOR COMPOUNDS: ALDEHYDES AND ALCOHOLS FROM LEUCINE BY MICROENCAPSULATED CELL-FREE EXTRACTS OF STREPTOCOCCUS LACTIS VAR. MALTIGENES

Cell-free extract (CFE) from Streptococcus lactis var. maltigenes was examined for its ability to catalyze the formation of 3-methyl butanal and 3-methyl butanol from leucine as affected by pH, substrate concentration and gradual addition of NADH. Pseudo Km and vmax, determined for CFE were 6.5 × 10-3 M and 7.1 μmol/liter-min when 3-methyl butanal was measured and 9.4 × 10-3 M and 2.4 μmol/liter-min for 3-methyl butanol. The CFE, leucine and cofactors were encapsulated in milkfat and incubated for 40 h at 22°, Rate of formation and final concentration of 3-methyl butanal and 3-methyl butanol were related to substrate concentration, indicating that flavor intensity can be controlled with encapsulated enzymes by limiting substrate concentration. Diffusion of 3-methyl butanol and acetic acid from microcapsules was affected by concentration and their lipophilic/hydrophilic character. Altering the pH of fluid surrounding microcapsules containing CFE had limited effect on the internal pH, as indicated by enzyme probes, demonstrating that capsules provide an environment isolated from the bulk phase of foods.

L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

Volatile Compounds Produced in Ground Muscle Tissue of Canary Rockfish (Sebastes pinniger) Stored on Ice

Volatile compounds associated with the progressive degradation of ground muscle tissue of canary rockfish (Sebastes pinniger) stored on ice were determined by combined gas–liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, n-propyl alcohol, acetaldehyde, propionaldehyde, acetone, ethyl alcohol, 2- and 3-pentanone, diacetyl, hexanal, 1-pentene-3-ol, 3-methyl-1-butanol, acetoin, trimethylamine, and dimethylamine. The following compounds were tentatively identified: propenal, octenal or octadienal, butyraldehyde, 3-methyl butanal, 2-butanone, and methyl vinyl ketone.

Investigation of the mechanism of vapour-phase dehydration of isopentene alcohols in the presence of acid catalysts

1. 1. The ease of dehydration of isopentene alcohols with acid catalysts decreases in the sequence 2-methyl 3-buten-2-ol>3-methyl 2-buten-1-ol>3-methyl 3-buten-1-ol The activity of catalysts in this reaction decreases in the following sequence: aluminosilicate>alumina>calcium phosphate . 2. 2. The low isoprene yield during dehydration of 3-methyl 3-buten-1-o1 in the presence of alumina and aluminosilicate is due to the cyclization of alcohol to β-methyltetrahydrofuran and subsequent splitting of oxacyclane to formaldehyde and isobutene. In the presence of tri-calcium phosphate which slightly accelerates cyclization, a high isoprene yield is obtained. 3. 3. The following reactions accompany dehydration of isopentene alcohols: isomerization of 3-methyl 2-buten-1-ol to isovaleric aldehyde, allyl re-grouping of 2-methyl 3-buten-2-ol to 3-methyl 2-buten-1-ol and 3-methyl 3-buten-1-ol and polymerization of isoprene. Alumina and aluminosilicate are more active in these reactions than calcium phosphate.

Essential Oils from the Queensland Flora. Part XVI. Eucalyptus microcorys.

SINCE the extensive investigations of Baker and Smith on the essentialnoils of the Australian Eucalypts, very little work has been donenon these important oils. Meanwhile great advance has been made innterpene chemistry and chemical technique and it was thought that oilsnfrom some of the more abundant species of Eucalypts could, withnadvantage, be sulbjected to a more thorough investigation.The essential oil of Eucalyptus microcorys was chosen as it growsnabundantly in southern Queensland, where it is cut for its timber knownnas Tallowwood.The essential oil was a light-yellow mobile oil, smelling strongly ofncineol.Baker and Smith recorded the presence of pinene, cineol, volatilenaldehydes and sesquiterpenes. We have confirmed these observationsnand also have shown the presence of isoamyl isovalerate, borneol,nbornyl acetate, and butyrate, and have proved the volatile aldehydenpresent to be isovaleric aldehyde. Also a small quantity of a carbonylncompound was isolated but not identified. n n

Attempted Synthesis of Terpene-like Substances from Isovaleraldehyde. I

Attempted Synthesis of Terpene-like Substances from Isovaleraldehyde. I

On Some Reactions of Isovaleraldehyde

On Some Reactions of Isovaleraldehyde