Nucleotide sequence of actinidin, a kiwi fruit protease

Podivinsky, Ellen; Forster, R. L. S.; Gardner, Richard C.

doi:10.1093/nar/17.20.8363

Cited by 41 publications

(27 citation statements)

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“…6). The amino acid sequence of EP-CI also exhibited high degrees of homology with those of other Cys endopeptidases reported to date, such as barley EP-B (60%) (Koehler and Ho, 1990), barley aleurain (43%) (Rogers et al, 1985), papain (46%) (Cohen et al, 1986), and actinidin (52%) (Podivinsky et al, 1989). Therefore, we postulate that EP-CI of French bean plants is a Cys endopeptidase of a type closely related to SH-EP, which is mainly expressed in cotyledons of germinating V. mungo seeds.…”

Section: Discussionmentioning

confidence: 75%

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

et al. 1993

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38: 1988-1995). In the present studies, one of the major endopeptidase forms, designated EP-C1, was purified as a 34-kD polypeptide from pods of maturing French bean fruits. EP-C1 was found to be immunologically distinguished from other forms in extracts from pods, but homologous to SH-EP, the major cysteine endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (W. Mitsuhashi, T. Minamikawa 119891 Plant Physiol89: 274-279). The leve1 of endopeptidase that reacted with the antiserum to EP-C1 increased in pods as the fruit maturation proceeded. EP-C1 was also immunologically detected in stems of French bean plants bearing fruits of later maturation stages. Protein immunoblotting showed that a 34-kD polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from 7-to 9-d cotyledons of germinating French bean seeds. In addition, two other polypeptides with slightly higher molecular masses were observed in extracts from 3-to 5-d cotyledons. We suggest that these two polypeptides are intermediates involved in posttranslational processing of EP-C1. RNA blot hybridization with EP-C1 cDNA as a probe showed that EP-C1 mRNA occurred in pods of fruits at later maturing stages and also in cotyledons of 3-to 7-d germinating seeds.

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Section: Discussionmentioning

confidence: 75%

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

et al. 1993

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“…Figure 1 shows the chimeric genes that were constructed and transformed into tobacco. The actinidin derivatives were produced, as described in "Materials and Methods," from the plasmid KIWI450 (Podivinsky et al, 1989), which contains a full-length actinidin cDNA. Two oligonucleotides, including one designed against the sequence around the junction of the CTPP with mature actinidin, were used in PCRs to generate two actinidin sequences.…”

Section: Protease Assaysmentioning

confidence: 99%

“…Possible solutions to the latter problem have emerged from the isolation and characterization of cDNAs encoding actinidin from kiwifruit (Praekelt et al, 1988;Podivinsky et al, 1989) and their comparison with cDNAs encoding other related Cys proteases. These Cys proteases were found to be encoded as preproproteins, with a putative ER-targeting signal and a large conserved NTPP.…”

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confidence: 99%

“…Chimeric genes were constructed to encode the fulllength preproprotein (NAC = NTPP-actinidin-CTPP) and the N-actinidin proprotein (NA = NTPP-actinidin) as shown in Figure 1. The actinidin gene from pKIWI450 (Podivinsky et al, 1989) was first transferred to pBluescript KS-(Stratagene) as an EcoRI-BamHI fragment, forming pWPlO0. The oligonucleotide primers 5'-GGGTCTAGAC-CATGGGTTTGCCCAAATCC-3' and 5'-GGGCCGCGGC-TAGTTGTTGTACTTGACGGG-3' were used in a PCR to generate an actinidin fragment (NA), which encodes a protein lacking the CTPP, from pWP100.…”

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confidence: 99%

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Correct Processing of the Kiwifruit Protease Actinidin in Transgenic Tobacco Requires the Presence of the C-Terminal Propeptide

et al. 1995

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A 35s cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-leve1 accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cieave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing.

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“…Actinidin consists of 220 amino acids with an apparent molecular weight of about 23.0 kDa (Kamphuis et al, 1985). Its complete amino acid sequence (Carne et al, 1978), the nucleotide sequence of the corresponding gene (Podivinsky et al, 1989), and the three dimensional structure (Varughese et al, 1992) have been determined.…”

Section: Introductionmentioning

confidence: 99%

Inactivation Kinetics and Structural Changes of High Pressure Treated Actinidin

Alexandrakis¹,

G.Katsaros²

2017

IJAST

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Plant sulfydryl proteases such as actinidin are mainly used as meat tenderisers, milk clotting agents for the development of novel dairy products and proteolytic agents in biotechnological procedures. The main issue with regards their application is the method for the control of their enzymatic activity after the desired extent of proteolysis, considering that thermal treatment is not applicable. High Pressure (HP) processing could be applied for the enzyme activity regulation.Actinidin, extracted from kiwi fruit (Hayward var., A. chinensis), was purified using anion-exchange and gel filtration chromatography. After purification procedure, only one single peak was finally received for this enzyme corresponding to a protein of molecular mass of 24 kDa with an isoelectric point equal to or lower than 3.5. The effect of HP (500-900MPa and 50-70°C) on the remaining activity of purified actinidin in phosphate buffer solution (pH 6.0) was studied. Inactivation of purified actinidin was described by a first-order kinetic model both for thermal and HP treatment at the studied processing conditions. Aiming at an optimal process design, a composite mathematical model, which describes the actinidin inactivation rate as a function of pressure and temperature conditions, taking into account the dependence of both activation energy and activation volume on the applied pressure and temperature respectively, was used.HP-induced conformational changes of the purified actinidin were also investigated using Circular Dichroism spectroscopy (CD). A direct comparison of the CD results for the treated and the untreated enzymes reveals that reversible changes were depicted in the far-and near-UV. It is suggested that the exposure to HP may affect the linkage between thiolate ion of cysteine group and positively charged nitrogen of the imidazole group of the active site of actinidin leading to the reduction of the enzyme activity.

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Nucleotide sequence of actinidin, a kiwi fruit protease

Cited by 41 publications

References 1 publication

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

Correct Processing of the Kiwifruit Protease Actinidin in Transgenic Tobacco Requires the Presence of the C-Terminal Propeptide

Inactivation Kinetics and Structural Changes of High Pressure Treated Actinidin

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