The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C

Clarke, Joyce; Shannon, Leland M.

doi:10.1016/0005-2795(76)90186-0

Cited by 82 publications

(26 citation statements)

References 15 publications

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“…In the horseradish root, HRP is found as a family of isoenzymes which appear to be the result of both multiple genes (Fujiyama et al, 1988) and different degrees of posttranslational modification with carbohydrate (Aibara et al, 1981). The most abundant isoenzyme is HRP C, which has a molecular mass of 44 kDa, a known amino acid sequence of 308 residues (Welinder, 1979) and, in addition to the iron (111) protoporphyrin IX active centre, has eight N-linked carbohydrate chains (Clarke and Shannon, 1976) and possibly one or two calcium ions. Although there is no X-ray structure available, there is a model, based on the sequence homology of HRP to the cytochrome-c peroxidase (Welinder, 1985).…”

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confidence: 99%

A step towards understanding the folding mechanism of horseradish peroxidase

Pappa

Cass

1993

European Journal of Biochemistry

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The guanidinium chloride denaturatiodrenaturation of the holo-and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies.A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed.Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca". The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca" and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Caz+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex.Horseradish peroxidase (HRP) has been extensively investigated over many years and has found widespread application in colorimetric test strips, as a label in both antibody and DNA probe methods and as a component of enzyme electrodes. In the horseradish root, HRP is found as a family of isoenzymes which appear to be the result of both multiple genes (Fujiyama et al., 1988) and different degrees of posttranslational modification with carbohydrate (Aibara et al., 1981). The most abundant isoenzyme is HRP C, which has a molecular mass of 44 kDa, a known amino acid sequence of 308 residues (Welinder, 1979) and, in addition to the iron (111) protoporphyrin IX active centre, has eight N-linked carbohydrate chains (Clarke and Shannon, 1976) and possibly one or two calcium ions. Although there is no X-ray structure available, there is a model, based on the sequence homology of HRP to the cytochrome-c peroxidase (Welinder, 1985).The catalytic mechanism of HRP has been well studied and the intermediate states (compounds 0, I and 11) characterised (Jones and Dunford, 1977;Adediran and Dunford, 1983; Van Waart, 1989, 1992;Ator and Ortiz de Montellano, 1987 A detailed analysis of the structure and function of H W would be aided by introducing point mutations and studying their effects on the catalytic activity and binding affinity of the substrates. Successful experiments with other haemoproteins expressed in Escherichia coli (Fishel et al., 1987, Phillips et al., 1990) encouraged us to express the synthetic gene for HRP, developed by Ortlepp et al. (1989), in E. coli. The E. coli system would not only facilitate mutagenesis work but would also produce non-glycosylated recombinant HRP, providing a possible starting material for X-ray crystallograAlthough HRP w...

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confidence: 99%

A step towards understanding the folding mechanism of horseradish peroxidase

Pappa

Cass

1993

European Journal of Biochemistry

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“…Attempts to purify ASC peroxidase by affinity chromatography with concanavalin A were unsuccessful because the enzyme would not bind under conditions which result in binding of horseradish peroxidase (4).…”

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Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

Da¹,

Fj²,

Sa³

et al. 1987

Plant Physiol.

124

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All aerobic biological systems, including N2-fixing root nodules, are subject to 02 toxicity that results from the formation of reactive intermediates such as H202 and free radicals of 02. H202 may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN Oxygen plays a critical but incompletely understood role in the metabolism of N2-fixing root nodules. Nodule function requires a delicate balance between respiratory 02 demand and the hazards of 02 toxicity. These hazards arise from the 02 sensitivity of nitrogenase and from the production of reactive intermediates such as H202 and superoxide free radicals (02*). A key defense against 02 toxicity is superoxide dismutase, an enzyme which has been detected in many aerobic organisms, including nodule host cells (21) and bacteroids (6). The superoxide dismutase reaction produces H202 which can also be damaging and must be removed through the action of catalase or peroxidase. Although catalase is present in soybean nodules (11,21), most peroxide removal probably occurs through a coupled series of oxidation-reduction reactions involving ASC3 and glutathione (5, Fig. 1 content of nodules and the activity of the first two enzymes in this system are positively correlated with nitrogenase activity and leghemoglobin content during the early stages of nodule development (5). ASC peroxidase and DHA reductase activities are not present in isolated bacteroids, thus implying these enzymes are of plant origin (5).All three of the enzymes in this system in chloroplasts have been purified and characterized (1,12,14), but very little information is available on the corresponding enzymes in root nodules. A peroxidase from soybean root nodules was characterized by Puppo et al. (20), but the effectiveness of ASC as an electron donor was not investigated. Legume root nodules reportedly contain numerous peroxidase isozymes (16), but this may be misleading because leghemoglobins are capable of very high pseudo-peroxidase activity in the presence of appropriate reductants (20).We present evidence in this report of the presence of ASC peroxidase and associated enzymes in nine species of legumes and in red alder (Alnus rubra). ASC peroxidase from soybean root nodules was purified to near homogeneity and characterized with regards to substrate specificity, substrate affinity, absorption spectra, and effects of inhibitors. The enzyme is compared with other peroxidases, especially the ASC peroxidase from spinach chloroplasts.

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“…5) (5,20,25). We have established here that the environmentally induced heritable difference in mol wt between corresponding peroxidases and corresponding acid phosphatases in the L and S flax genotrophs is due to the presence of additional mannose residues on the oligosaccharide chains ofthe S isozymes compared to the corresponding L isozymes.…”

Section: Methodsmentioning

confidence: 79%

Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with α-Mannosidase

Gaudreault¹,

Tyson²

1988

Plant Physiol.

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ABSTRACIIn the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with a-mannosidase, the difference in mobility was eliminated. Treatment with a-fucosidase and j8-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-O-N-acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes.Durrant (8,9) Rm value of the L type (11, 13). These observations suggest that the Rm shifts are the result of posttranslational modifications since it is unlikely that changes across all possible structural gene loci for these enzymes would yield unidirectional Rm shifts. The absence ofco-dominance in F, hybrids ofL and S for all isozymes of peroxidase (24) also points to differences in posttranslational processing. An obvious common site for posttranslational modification of both the peroxidase and acid phosphatase isozymes in genotrophs L and S is their carbohydrate moiety since they all are glycoproteins (14). This is further supported by the recent observations that corresponding L and S isoperoxidases can be chromatographically separated from one another on columns of Sepharose-bound Con A (15) but not by isoelectric focusing nor can they be distinguished from one another by immunochemical methods (16).In this report, we present results from investigations into the structure of the carbohydrate moiety of purified L and S peroxidase isozymes using column chromatography on several Sepharose-bound lectins with different sugar affinities and the effect of various glycosidase digestions on the Rm difference between L and S peroxidase and acid phosphatase isozymes. The anodic peroxidase pattern in several other flax genotypes was also examined along with the effect of a-mannosidase treatment on their migration on PAGE. Our results suggest that the Rm differences between L and S glycoenzymes are due to higher levels of mannosylation of these proteins in the genotroph S. MATERIALS AND METHODS

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The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C

Cited by 82 publications

References 15 publications

A step towards understanding the folding mechanism of horseradish peroxidase

A step towards understanding the folding mechanism of horseradish peroxidase

Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with α-Mannosidase

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