Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide glucoside by an enzyme from ox brain

Ceramide glucoside (1-O-glucosido-2-N-acyl-sphingosine) was hydrolysed to ceramide (N-acyl-sphingosine) and glucose by β-glucosidase from ox brain. The reaction was stimulated by the non-ionic detergent, Triton X-100, or by the anionic detergents, cholate or taurocholate. It was not reversible, had optimum pH5·0 (with acetate buffer) or 5·6 (with pyridine buffer), had K_m 1·8×10⁻⁴m and was inhibited by δ-gluconolactone and sphingosine, but not by ceramide or palmitic acid.     

Electrophoretic and other studies on haem pigments from Rhodopseudomonas palustris: Cytochrome 552 and cytochromoid c

1. Cytochrome 552 and cytochromoid c were extracted from Rhodopseudomonas palustris cells, purified and obtained in crystalline form. 2. Extinction ratios and amino acid compositions of the two pigments are reported. 3. When subjected to starch-gel electrophoresis in borate buffer, pH8·8, each pigment migrated towards the cathode; oxidized cytochromoid c migrated more rapidly than its reduced form. 4. By a determination of electrophoretic mobilities in buffers of I0·1 by using the moving-boundary method, the isoelectric point of cytochrome 552 was found to be at pH10·6 and that of cytochromoid c at pH9·7. 5. As obtained, cytochrome 552 was non-autoxidizable; cytochromoid c was autoxidizable but became considerably less so on alkaline treatment. 6. Discussion of the results includes a consideration of the isoelectric points of the pigments in terms of their amino acid composition.     

Effects of pH and temperature on the wild-type and a mutant form of Neurospora glutamate dehydrogenase

1. We confirm the observation of Bürk (1965) that Neurospora crassa NADP-linked glutamate dehydrogenase normally exists in an inactive form below pH7·0 and in a fully active form above pH8·0 in either tris or orthophosphate buffer. At pH7·4 the enzyme is about half activated at 25°. 2. The variety of the enzyme produced by the mutant am^2l shows a similar behaviour except that the transition is shifted about one pH unit in the alkaline direction. 3. The am^2l enzyme has previously been reported to be activated by brief warming to 30° in phosphate buffer at pH8·0. The wild-type enzyme shows a similar effect at pH7·0. In tris buffer this effect is much less pronounced. 4. The am^2l enzyme is extremely unstable at 47° at pH7·0; its stability is somewhat greater at lower pH, and is markedly increased by increasing the pH in the range 7·0–8·7. The wild-type enzyme also shows an indication of a stability minimum at pH7·0, but a temperature of 60° is needed for a measurable rate of inactivation. 5. The inactive form of the enzyme is much more subject to thermal irreversible denaturation than is the active form.     

Thermal denaturation in acidic solutions of double-helical ribonucleic acid from virus-like particles found in Penicillium chrysogenum. A spectrophotometric study

1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of pK3.9 compared with pK2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε_(P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol<sup>−1</sup>·cm<sup>−1</sup> depending on pH, compared with 33501·mol<sup>−1</sup>·cm<sup>−1</sup> at pH7. In contrast ε<sub>(P)</sub> at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values.     

Combinations of Intervention Treatments Resulting in 5-Log10-Unit Reductions in Numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 Organisms in Apple Cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log₁₀ units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log₁₀ units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log₁₀-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log₁₀-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log₁₀-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

The thermal polymerization of orosomucoid

1. Orosomucoid was prepared from the urine of a nephrotic patient and polymerized by heating it in a range of salt concentrations at pH4·1. 2. Heating at low ionic strengths produced a `chain' polymer of indefinite length but having the same width as the diameter of the monomer (5·0nm.). Similar treatment in high ionic strengths also produced a spherical (`ball') polymer of limited diameter (14·8nm.). 3. The size and shape of both polymers were determined from ultra-centrifuge, gel-filtration and electron-microscope results. The results suggest that eight monomer units condense to form the ball polymer. 4. Heating orosomucoid at pH1·8 hydrolysed the N-acetylneuraminic acid off the molecule; only chains could then be formed, even in high ionic strengths. 5. Both polymers were stable under normal conditions but could be depolymerized in 3m-guanidine hydrochloride. The monomer could be repolymerized on heating: the `chain monomer' only formed chains at all ionic strengths, but the `ball monomer' was indistinguishable from the original monomer in its immunological properties and polymerization reaction.     

Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn²⁺, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Enzymes in cancer: Asparaginase from chicken liver

1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (K_m) at 37° and pH8·5 were 6·0×10⁻⁵m and 7·2×10⁻⁵m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with K_i approx. 5·6×10⁻⁴m. In mice it produced an anti-lymphoma effect, as reported previously.     

Presence, induction and possible role of glucose 6-phosphatase in mammalian pancreatic islets

1. Pancreatic islets from several mammalian species were investigated for hydrolytic activity towards glucose 6-phosphate. Both the total phosphatase activity towards this substrate and the proportion cleaving glucose 6-phosphate in preference to β-glycerophosphate varied widely between species. In pancreatic-islet homogenates prepared from mice and guinea pigs there was a higher rate of liberation of P_i at pH6·7 from glucose 6-phosphate than from β-glycerophosphate. In these two species cortisone treatment enhanced the enzyme activity towards glucose 6-phosphate but not that towards β-glycerophosphate. Simultaneous injections of ethionine or puromycin blocked this stimulating effect of cortisone. 2. With whole homogenates of mouse pancreatic islets, inverse plots of the relationship between glucose 6-phosphate concentration and enzyme activity suggested the simultaneous action of two enzymes with different K_m values. After fractionation of islets from obese–hyperglycaemic mice by differential centrifugation, one of these enzymes could be shown to be localized in the microsome fraction. It had K_m for glucose 6-phosphate about 0·5mm and optimum pH6·7. It split glucose 6-phosphate in preference to β-glycerophosphate, glucose 1-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. Incubation of the microsomes at pH5·0 and 37° for 15min. decreased the enzyme activity by about 80%. Glucose was a potent inhibitor, the type of inhibition being neither strictly competitive nor non-competitive. It is suggested that the results indicate the presence of glucose 6-phosphatase in mammalian endocrine pancreas, and that this enzyme may play a role in the metabolic regulation of release of insulin.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Ozone Degrades into Hydroxyl Radical under Physiological Conditions : A Spin Trapping Study

Defining the reactants is a critical step towards elucidating the mechanism of ozone toxicity to biomembranes. To document ozone-induced HO·radicals, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide was used and the resulting spin adduct was monitored with electron spin resonance spectroscopy. Chelexed potassium phosphate buffer (10 millimolar and 0.2 molar) at pH 7.2 and 7.8 was exposed to ozone (1-40 microliters per liter) by directing a stream of ozone over the surface for 60 seconds. Under these conditions, no HO· was detected. Using 0.5 × 10⁻⁴ molar caffeic acid in phosphate buffer, strong DMPO·OH electron spin resonance signals were obtained, indicating HO· production. Air controls yielded no signal. High pH (7.8) enhanced signal strength. Furthermore, with sorbitol (0.4 osmolal final concentration), a net HO· signal loss of 28% was observed, while a carbon-centered sorbitol radical adduct appeared. Although HO· radicals were produced, no breakage of Daucus carota protoplast plasma membranes was observed nor were differences in membrane fluidity observed as determined by 5-doxyl stearic acid.     

Purification, properties and mechanism of action of a staphylolytic enzyme produced by Aeromonas hydrophila

1. An enzyme produced by Aeromonas hydrophila and capable of lysing Staphylococcus aureus cells was purified 180-fold by gel filtration and chromatography on columns of AG-50 W resin. 2. Physical measurements on the purified enzyme suggest that it is a small basic protein with an isoelectric point between pH9·0 and pH9·5. 3. Maximum lytic activity was obtained in 20mm-tris–glycine buffer, pH8·5, at 45°, with no detectable activity in the absence of a nitrogenous base. 4. The enzyme is active in the above buffer containing 1·5m-sucrose, and is useful for the preparation of protoplasts of Staphylococcus aureus. 5. Purified cell wall peptidoglycans of two strains of Staphylococcus aureus, differing in amino acid composition, were hydrolysed by the enzyme with the liberation of glycine oligopeptides, principally diglycine and triglycine. 6. Synthetic glycine oligopeptides larger than triglycine, but not polyglycine, were hydrolysed, as were a number of leucine-containing dipeptides and tripeptides, but no proteolytic activity could be demonstrated. 7. It is concluded that the enzyme is lytic towards Staphylococcus aureus because it splits the pentaglycine cross-links of the cell-wall peptidoglycan.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

Further observations on the production of amino acids by rat-liver mitochondria and other subcellular fractions

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0·25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn²⁺ (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0·9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0·9% potassium chloride or 0·25m-sucrose was similar (q_{amino acid} 0·047 and 0·042 respectively aerobically). After freezing-and-thawing q_{amino acid} values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg²⁺ and Zn²⁺ being the most effective in tris–hydrochloric acid buffer. In phosphate buffer Mg²⁺ and Mn²⁺ had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70–80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from α-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris–hydrochloric acid but not in phosphate buffer, α-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21–24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25° and 40°. The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of ¹⁴C-labelled algal protein or denatured mitochondrial protein.     

Control of Flavor Development in Wine during and after Malolactic Fermentation by Oenococcus oeni

During malolactic fermentation in wine by Oenococcus oeni, the degradation of citric acid was delayed compared to the degradation of malic acid. The maximum concentration of diacetyl, an intermediary compound in the citric acid metabolism with a buttery or nutty flavor, coincided with the exhaustion of malic acid in the wine. The maximum concentration of diacetyl obtained during malolactic fermentation was strongly dependent on the oxygen concentration and the redox potential of the wine and, to a lesser extent, on the initial citric acid concentration. The final diacetyl concentration in the wine was also dependent on the concentration of SO₂. Diacetyl combines rather strongly with SO₂ (K_f = 7.2 × 10³ M⁻¹ in 0.1 M malate buffer [pH 3.5] at 30°C). The reaction is exothermic and reversible. If the concentration of SO₂ decreases during storage of the wine, the diacetyl concentration increases again.