Eitan Harel

Polyphenol oxidases in plants

Abstract Recent progress in the study of plant polyphenol oxidases is critically reviewed. Two main groups are recognized: the catecholoxidases and the laccases. Their purification, subcellular location and protein properties are described. Attention is also given to their activation and induction, their function and evolution.

Assay of catechol oxidase—a critical comparison of methods

Abstract Manometric, polarographic, chronometric and spectrophotometric methods for the determination of catechol oxidase activity were critically compared. Where possible, product formation, disappearance of substrate and ratios between oxygen uptake and substrate disappearance were determined. Instability of products and secondary reactions interfered in some of these determinations. Initial rates measured polarographically were eight to twelve times higher than those obtained by the spectrophotometric and chronometric methods and up to 30 times greater than those obtained manometrically. The polarographic method is recommended as the most convenient and accurate method for determining catechol oxidase activity.

Import, Targeting, and Processing of a Plant Polyphenol Oxidase

A tomato (Lycopersicon esculentum L.) gene encoding a precursor of polyphenol oxidase(PPO) was transcribed and translated in vitro. The import, targeting, and processing of the [35S]methionine-labeled precursor protein (pPPO) were studied in isolated chloroplasts. The protein was routed to the thylakoid lumen in two steps. The 67-kD precursor was first imported into the stroma in an ATP-dependent step. It was processed to a 62-kD intermediate by a stromal peptidase. Translocation into the lumen was light dependent and involved processing of the 62-kD to the 59-kD mature form. The mature polypeptide was soluble in the lumen and not bound to thylakoids. This two-step targeting pattern was observed in plastids from a variety of plants including pea (Pisum sativum L.), tomato, and maize (Zea mays L.). The ratio between the intermediate and mature forms observed depended on the plant species, leaf age, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. Furthermore, low concentrations of Cu2+ (1–5 [mu]M) markedly inhibited the first import step. Tentoxin specifically inhibited pPPO import, leaving the precursor bound to the envelope membrane. The two-step routing of pPPO into chloroplasts, typical of thylakoid lumen proteins, is consistent with the two-domain structure of the transit peptide and appears to be a feature of all plant PPO genes isolated so far. No evidence was found for unorthodox routing mechanisms, which have been suggested to be involved in the import of plant PPOs. The two-step routing may account for some of the multiplicity of PPO observed in vivo.

Purification and multiplicity of catechol oxidase from apple chloroplasts

Abstract Catechol oxidases were extracted from subcellular fractions of apples. Triton X-100 was efficient in extracting the enzymes from chloroplasts and digitonin from mitochondria. The enzymes present in the chloroplast extract were separated by starch gel electrophoresis into three bands which corresponded to fractions obtained by chromatography on DEAE-cellulose columns. The fractions were differentiated according to K m , substrate specificity and sensitivity to inhibitors. A three-hundred-fold purification of the major fraction was obtained. The evidence for a real multiplicity and its possible implications are discussed.

Catechol oxidase from green olives: Properties and partial purification

Abstract Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.

Partial purification and properties of catechol oxidases in grapes

Abstract Grapes contain a high level of catechol oxidase activity. The enzyme is located in a particulate fraction of the cell, apparently in the plastids. It resembles catechol oxidases from other fruits in many aspects but differs in its relatively high activity towards caffeic and p-hydroxycinnamic acids and in its high affinity towards the latter. The enzyme is relatively insensitive to most inhibitors of catechol oxidase. The enzyme was solubilized with 1% Triton X-100 and purified 115 fold by ammonium sulphate fractionation and column chromatography. The enzyme from grapes, like other catechol oxidases, can be resolved into several fractions by gel electrophoresis.

Evidence for conformational changes in grape catechol oxidase

Abstract A rapid, 4–10-fold, activation of grape catechol oxidase by a short exposure to acid pH or urea is demonstrated. Activation was either reversible or irreversible, depending on length and type of treatment. The change in activity of the enzyme is due primarily to an increase in V max , while the affinity for 4-methylcatechol decreases and that for O 2 increases. Activation occurs in intact chloroplasts as well as in a partially purified enzyme preparation. Activation was apparently due to conformational changes in the enzyme. O 2 concentration appeared to control enzyme activity, presumably by an O 2 induced conformational change. Irreversible activation was accompanied by changes in the electrophoretic mobility of the enzyme.

Purification and properties of laccase from Botrytis cinerea

Abstract The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its Km for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.

Pectin, a second inducer for laccase production by Botrytis cinerea

Abstract Pectin acts as a second inducer of extracellular laccase formation by Botrytis cinerea , in the presence of a phenolic substance as a first inducer, but pectin alone fails to induce enzyme formation. The possible advantages of this mechanism for the fungus during the process of infection and overcoming host resistance are discussed.

Interconversion of sub-units of catechol oxidase from apple chloroplasts

Abstract The three fractions of catechol oxidase previously reported from apple chloroplasts were shown to result from various degrees of aggregation of sub-units of the same enzyme. This was demonstrated by gel filtration on columns and by thin layer gel filtration in Sephadex. Interconversion between the fractions takes place during storage at 2–4° and is enhanced by various treatments. The possible existence of such interconversions in vivo and their physiological significance are discussed.