Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

Hughes, Monica A.; Dunn, Michael

doi:10.1007/bf00021030

Cited by 36 publications

(29 citation statements)

References 24 publications

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“…volved in cyanogenesis (10,14,21,24). At pH 5.0, the AH isozymes displayed insignificant differences in their Km and Vmax values toward their endogenous substrate amygdalin (Table II).…”

Section: Resultsmentioning

confidence: 98%

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

Li¹,

Swain²,

Poulton³

1992

Plant Physiol.

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In black cherry (Prunus serotina Ehrh.) seed homogenates, amygdalin hydrolase (AH) participates with prunasin hydrolase (PH) and mandelonitrile lyase in the sequential degradation of (R)-amygdalin to HCN, benzaldehyde, and glucose. Four isozymes of AH (designated AH I, I', II, II') were purified from mature cherry seeds by concanavalin A-Sepharose 4B chromatography, ion-exchange chromatography, and chromatofocusing. All isozymes were monomeric glycoproteins with native molecular masses of 52 kD. They showed similar kinetic properties (pH optima, K., Vmax) but differed in their isoelectric points and N-terminal amino acid sequences. Analytical isoelectric focusing revealed the presence of subisozymes of each isozyme. The relative abundance of these isozymes and/or subisozymes varied from seed to seed. Three isozymes of PH (designated PH I, Ila, and lIb) were purified to apparent homogeneity by affinity, ion-exchange, and hydroxyapatite chromatography and by nondenaturing polyacrylamide gel electrophoresis. PH I and PH lIb are 68-kD monomeric glycoproteins, whereas PH lla is dimeric (140 kD). The N-terminal sequences of all PH and AH isozymes showed considerable similarity. Polyclonal antisera raised in rabbits against deglycosylated AH I or a mixture of the three deglycosylated PH isozymes were not monospecific as judged by immunoblotting analysis, but also cross-reacted with the opposing glucosidase. Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen.the sequential action of the enzymes AH2, PH, and MDL (12,29). One of the complexities of the Prunus cyanogenic system yet to be satisfactorily unraveled is the microheterogeneity shown by these catabolic enzymes. Earlier studies (13,14,29) established that AH and PH occur in cherry homogenates as two and three isozymes, respectively, whose existence was not attributable to partial proteolysis during isolation. The nature and physiological significance of this multiplicity might best be approached by N-terminal sequencing followed by construction and screening of a black cherry cDNA expression library. As with other cyanogenic species, large-scale cyanogenic glucoside catabolism in P. serotina occurs only after tissue disruption, implicating some crucial compartmentation of amygdalin and its catabolic enzymes in undamaged seeds. However, such spatial regulation of cyanogenesis remains poorly understood (27,28).In this paper, we describe how previous purification procedures have been improved to achieve the isolation of four AH and three PH isozymes for use in N-terminal sequencing. Monospecific polyclonal antibodies have been generated against AH and PH for future use in immunocytochemical localization studies and screening of expression libraries. Additionally, to gain further insight into the multiplicity shown by AH, we have devised a simple protocol involving affinity chromatog...

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“…volved in cyanogenesis (10,14,21,24). At pH 5.0, the AH isozymes displayed insignificant differences in their Km and Vmax values toward their endogenous substrate amygdalin (Table II).…”

Section: Resultsmentioning

confidence: 98%

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

Li¹,

Swain²,

Poulton³

1992

Plant Physiol.

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“…The cyanogenic j3-glucosidase (linamarase) of white clover is a homodimer with a subunit molecular mass of 62,000 Mr. It is the major soluble high-mannose asparagine-linked glycoprotein in young leaves, where it can represent up to 5 per cent of the total soluble protein (Hughes & Dunn, 1982). The antibiotic, tunicamycin, prevents linamarase synthesis but no precursor polypeptides have been found in vivo.…”

Section: Biochemistrymentioning

confidence: 99%

“…Heterozygotes, (Li Ii) produced intermediate niRNA levels in young leaf tissue. The presence of low but measurable levels of linamarase homologous mRNA in Table 1 The quantitative inheritance of HCN production (after Corkil, 1941) (Hughes & Dunn, 1982;Collinge & Hughes, 1982a;Hughes et at., 1985;Kakes & Eettink, 1985). Because small changes in the protein primary structure may result in major differences in the antigenic properties, it is possible for linamarase cDNA to show homology to a non-cyanogenic /3-glucosidase mRNA produced at low levels in these tissues.…”

Section: Molecular Geneticsmentioning

confidence: 99%

The cyanogenic polymorphism in Trifolium repens L. (white clover)

1991

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The cyanogenic polymorphism in white clover is controlled by alleles of two independently segregating loci. Biochemical studies have shown that non-functional alleles of the Ac locus, which controls the level of cyanoglucoside produced in leaf tissue, result in the loss of several steps in the biosynthetic pathway. Alleles of the Li locus control the synthesis of the hydrolytic enzyme, linamarase, which is responsible for HCN release following tissue damage. Studies on the selective forces and the distribution of the cyanogenic morphs of white clover are discussed in relation to the quantitative variation in cyanogenesis revealed by biochemical studies. Molecular studies reveal considerable restriction fragment length polymorphism for linamarase homologous genes.

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“…The active enzyme is a homodimer, with a subunit molecular weight of Mr 62,000 and is ionically bound within the cell (Hughes and Dunn, 1982). It is tissue specific and synthesised during early leaf development.…”

Section: Introductionmentioning

confidence: 99%

“…It has been shown that these non-functional null alleles (Ii) result in the absence of active linamarase protein and of antigenically related inactive protein (Hughes and Dunn, 1982;Dunn, 1984). A plant with an inherited reduced level of linamarase activity has been identified (Maher and Hughes, 1973) and this paper reports on a further immunochemical and genetical study of another element which is shown to control the amount of linamarase protein synthesised by leaf tissue.…”

Section: Introductionmentioning

confidence: 99%

A regulatory element controlling the synthesis of the cyanogenic β-glucosidase (Linamarase) of white clover

1985

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A variant form of white clover, which produces reduced levels of the cyanogenic j3-glucosidase (linamarase) activity, has been identified. The reduced level of linamarase activity is determined by an element which lies within 4 map units of the Li locus. The low level of linamarase activity in the variant plant and its progeny is due to a reduction in the rate of synthesis of linamarase in developing leaf tissue.

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Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

Cited by 36 publications

References 24 publications

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

The cyanogenic polymorphism in Trifolium repens L. (white clover)

A regulatory element controlling the synthesis of the cyanogenic β-glucosidase (Linamarase) of white clover

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