鹿児島大学リポジトリ

(1)

PROTEASE ACTIVITY IN PLANT TISSUES (V)

著者

UCHIKOBA Tetsuya, AMAKATSU Kousuke, KANEDA

Makoto

journal or

publication title

鹿児島大学理学部紀要. 数学・物理学・化学

volume

23 page range

139-145

別言語のタイトル

種々の植物組織のプロテアーゼ活性について (v)

URL

http://hdl.handle.net/10232/6479

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PROTEASE ACTIVITY IN PLANT TISSUES (V)

著者

UCHIKOBA Tetsuya, AMAKATSU Kousuke, KANEDA

Makoto

journal or

publication title

鹿児島大学理学部紀要. 数学・物理学・化学

volume

23 page range

139-145

別言語のタイトル

種々の植物組織のプロテアーゼ活性について (v)

URL

http://hdl.handle.net/10232/00004001

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Rep. Fac. Sci., Kagoshima Univ., (Math., Phys. & Chem.) No. 23, p. 139-145, 1990.

PROTEASE ACTIVITY IN PLANT TISSUES (V)

Tetsuya UCHIKOBA, Kousuke AMAKATSU, Makoto KANEDA*

(Received Sep. 3, 1990)

Abstract

Extracts from various plants were examined for protease activity. High caseinolytic activity was found in the extracts of bulb of tulip, Tulipa ge∫neriana L., and Paper mulberry, Brou∫∫onetia papyrifera Vent.

High 4-nitroanilide hydrolytic activity was found in the extracts of Chayote, Sechium edule

Swartz for Leu-PNA, and Hananira, Brodiaea multiflora Benth. for Ala-PNA.

Introduction Anumberofplantproteaseshavebeenstudied,usuallyemphasizingtheproperties ofsuchwell-knowncysteineenzymesaspapain(l),ficin0?),andbromelain(3).In contrasttotheabovecysteineproteases,relativelylittleisknownaboutothertypesof proteasefromplantsources. As ingtest:su fV;:essortoourpreviouspaper(4-7),wedescribeheretheproteasescreen i｡usplants.Inadditiontheamin｡peptidaseactivity｡fplantsweredeter二 minedtofindanewplantenzymesource. Experimental Fruitsandcerealswerepurchasedfromgreengrocersandotherplantswerecol-lectedlocallyinKagoshimaprefecture.CaseinwasaproductofE.Merck,Darmstadt, WestGermany;Ala-PNA,Leu-PNAwasobtainedfromPeptideInstitute,Inc.,Osaka, Japan;OtherreagentswerepurchasedfromWakoPureChemicalIndustriesLtd. PreparationofSampleSolutionforCa∫einolyticA∫∫ay-A¥¥samplesweregroundinequal weightof0.02Mphosphatebuffer,pH7.3,inamortarandthehomogenateswerestir-redfor5minandfilteredthroughacottonclothorcentrifugedfor10minat3000×g. Theextractsweredilutedtothepointofappropriateconcentrationforassaywith 0.02Mphosphatebuffer,pH7.3. preparationofSampleSolutionfor4-NitroanilideHydrolyticActivityAssay-Solid(NEU^Sc^ wereaddedtotheextractsofsampleplantsto60%saturation.Afterstandingfor24h theresultingppt.wascollectedbycentrifugationandthendialyzedagainstwater. Thesefiltrateswereusedassamplesolution.

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140 _{Tetsuya Uchikoba, Kousuke Amakatsu, Makoto Kaneda}

A∫∫ay ofProtect∫｣-Proteolytic activity was measured by two methods.

Caseinolytic activity was assayed by method of Kunitz (#) , with casein as a substrate.

One ml of sample solution was preincubated for 10 min at 30 , and then added to 1 ml of

a solution of 1% (w/w) casein containing 0.02 M phosphate buffer, pH 7.3, at 300,

Af-ter incubation for 30 min the reaction was Af-terminated by the addition of 2 ml of 5%

trichloroacetic acid. After standing for 30 min at room temperature, the precipitate was

removed by filtration through Toyo filter paper No. 5C and the absorbancy at 280 nm of

the trichloroacetic acid-soluble peptides formed was determined with Hitachi

spec-trophotometer 100-60.

The rates of enzymatic hydrolysis of 4-nitroanilide substrates were followed

spec-trophotometrically in 0.1 M Tris-HCI buffer, pH 7.5, at 410 nm with the

spectrophot0-meter.

A unit of activity was defined as that amount which yielded 0.001 A280nm (0.001

A4iOnm) unit of change per min under the conditions mentioned above. The specific

activity is expressed as the number of enzyme units per 1 ml of Juice.

Results and Discussion

The results of the screening test are shown in Table 1, 2.

Caseinolytic activity was observed in several plants. The activity of the extracts

of bulb of tulip, Tulipa gesneriana L, and Paper mulberry, Broussonetia papyrifera Vent,

prom-inent in the sample tested. But the number of units were not so large compared with sarcocarp of melons (4, 7) or snake gourds (5, 6). All of them are cucurbitaceae family. Paper mulberry protease was confirmed to be cysteine protease by further investigation.

As this plant is Moraceae family, it is considered the cysteine protease group of Moraceae

like a ficin from fig, Ficus Carica L.

The extracts of thirteen plants hydrolyzed 4-nitroanilide. The highest activity

were Chayote, Sechium edule Swartz for Leu-PNA, and Hananira, Brodiaea multiflora Benth.

for Ala-PNA. But these activity units were not enough to study plant aminopeptidase.

References

1. Arnon, R. (1970) in Methods in Enzymology (Perlmann, G. E. & Lorand, L., eds.) Vol. 19, pp. 226-244, Academic Press, New York

2. Liener, I. E. & Friedenson, B. (1970) in Methods in Enzymology (Perlmann, G. E. & Lorand, L., eds.) Vol. 19, pp. 26ト273, Academic Press, New York

3. Murachi, T. (1970) in Methods in Enzymology (Perlmann, G. e. & Lorabd, L., eds.) Vol. 19, pp. 273-284, Academic Press, New York

4. Kaneda, M.t Yonezawa, H., & Tominaga, N. (1982) Rep. Fac. ScL, Kago∫hima Univ., (Math., Phys., & Chem.) No. 15, pp. 53-55

5. Kaneda, M., Uchikoba, T.f Furugen, K., & Tominaga, N. (1985) Rep. Fac. ScL, Kago∫hima Univ., (Math., Phys., & Chem.) No. 18, pp. 59-63

6. Uchikoba, T.f Izumi, S., Fukuda, T., Kaneda, M., & Tominaga, N. (1987) Rep. Fac. Sci., Kagoshima Univ. (Math., Phys., & Chem.) No. 20, pp. 77-79

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PROTEASE ACTIVITY IN PLANT TISSUES (V) 141

7. Uchikoba, T., Sata, I., Akiba, H., Ishihara, S., & Kaneda, M., (1988)Rep. Fac. ScL, Kago∫hima Univ. (Mach., Phys., & Chem.) No. 21, pp. 105-110

Kunitz, M.f (1947) / Gen. Phy∫iol. 30, 291

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142 Tetsuya Uchikoba, Kousuke Amakatsu, Makoto Kaneda

Table 1. Caseinolytic Activity of Extracts from Plant Tissues Plant Method of

Plant parts extraction

Akinotamurasou

(Salvia japonica Thunb.) Arechinogiku

(Erigeron banariensis L. E. linifolius Willd.)

Biito, Garden Beet

(Beta vulgaris L. var. rubra Maq.) Chikori, Endive

(Cichorium endivia L.) Churippu, Tulip

(Tulipa gesneriana L.) Gishigishi

(Rumex iaponicus Houttuyn) Hananira

(Brodiaea multiflora Benth.) Hariguwa,

(Cudrania tricuspadata Bureau) Hayatouri, Chayote

(Sechium edule Swartz) Hechima, Suakwa Towelgourd

(Luffa cylindrica Roem.) Hekusokazura

(Paedaria scandens Merrill) Hisakaki

(Eurya japonica Thunb) Hishi, Water Caltrops

(Trapa natans L. var. bispinosa Makino)

Hiyashinsu, Common Hyacinth (Hyacinthus orientalis L.) Inutade

(Polygonum Bzumei Meisn.)

Leaf

Whole

Bulb

Whole

Leaf Sarcocarp Sarcocarp Leaf Leaf, Fruit Sarcocarp Ext Ext Ext Ext 13 Ext 97 Ext Ext 63 Ext Ext Ext Ext 18 Ext Ext Ext 69 Ext 26

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(from the Table 1)

Kazinoki, Paper mulberry

(Broussonetia papyrifera Vent.) Kinkan, Kumguat

(Fortunella crassifolia Swingle) Kohzo

(Broussonetia Kazinoki Sieb.) Kusaichigo

(Rubus hirsutus Thunb.) Kusaniwatoko

(Sambucus chinensis Lindl.) Makomo, Manchuriam wild rice

(Zizania latifolia Turcz.) Nihonzuisen, Chinese sacred lily

(Narcissus tazetta var. chinensis Roem.) Okatoranoo

(Lysimachia clethroides Duby) Oniyuri, Tiger Lily

(Lilium lancifolium Thunb.) Udo, Udo

(Aralia cordata Thunb.) Wasabi, Wasabi

(Eutrema japonica (Mig.) Koidz.) Yabutsubaki, Common camellia

(Camellia japonica L.) Yamamomo

(Myrica rubra Sieb. et Zucc.) Yuzu, Yuzu

(Citrus junos Sieb. ex T. Tanaka)

Berry Leaf Fruit Leaf Leaf

Whole

Bulb

Whole

Stem

Whole

Sarcocarp Seed Sarcocarp Fruit 上川い上川い川は u X X X E E E 川は川は u 叩川 u X X X E E E 7 0 0 9 0 0 0 5 0 0 0 0 1 0 r: 0 Ext: Extract

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144 Tetsuya Uchikoba, Kousuke Amakatsu, Makoto Kaneda

Table 2. 4-Nitroanilide Hydrolytic Activity of Extracts from Plant Tissues Plant Activity (Units) Plant parts Leu-PNA Ala-PNA

Arechinogiku

(Erigeron banariensis L. E. linifolius

Willd.)

Biito, Garden Beet

(Beta vulgaris L. var. rubra Maq.) Chikori, Endive

(Cichorium endivia L.) Churippu, Tulip

(Tulipa gesneriana L.) Gishigishi,

(Rumex japonicus Houttuyn) Hananira

(Brodiaea multiflora Benth.) Hasu, East indian lotus

(Nelumbo nucifera Gaertn) Hayatouri, Chayote

(Sechium edule Swartz) Hechima, Suakwa Towelgourd

(Luffa cylindrica Roem.) Hishi, Water Caltrops

(Trapa natans L. var. bispinosa Makino) Hiyashinsu, Common Hyacinth

(Hyacinthus orientalis L.) Ichijiku, Fig

(Ficus Carica L.) Kinkan, kumguat

(Fortunella crassifolia Swingle) Kusaniwatoko

(Sambucus chinensis Lindl.) Kyohou

(Vitis vinifera L. `Kyohou') Makomo, Manchuriam wild rice

(Zizania latifolia Turcz.)

Whole

Bulb

Whole

Rhizome Fruit 167 Fruit Sarcocarp 10 Bulb 40 Sarcocarp 57 Fruit

Whole

Fruit

Whole

0 0 0 45 0 36 0 0 84 0 83 0 8 0 9 0 0 0 50

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(from the Table 2)

Myouga

(Zingiber mioga Rose.) Ninniku, Garlic

(Allium sativum L. forma pekinese Makino) Okatoranoo

(Lysimachia clethroides Duby) Oniyuri, Tiger Lily

(Lilium lancifolium Thunb.) Shiitake

(Lentinus edodes Sing.)

Toogan, White gourd

(B占nincasa cerifera Savi) Udo, Udo

(Aralia cordata Thunb.) Wasabi, Wasabi

(Eutrema japonica (Mig.) koidz.) Yabutsubaki, Common camellia

(Camellia japonica L.) Yuzu, Yuzu

(Citrus junos Sieb. ex T. Tanaka)

Whole

16 0 0 0 Fruit body 28 Sarcocarp 19 Stem

Whole

Sarcocarp Seed Fruit 0 0 0 5 0 0 0 28 9 0 0 0 0

鹿児島大学リポジトリ

PROTEASE ACTIVITY IN PLANT TISSUES (V)

著者

UCHIKOBA Tetsuya, AMAKATSU Kousuke, KANEDA

Makoto

journal or

publication title

鹿児島大学理学部紀要. 数学・物理学・化学

volume

23

page range

139-145

別言語のタイトル

種々の植物組織のプロテアーゼ活性について (v)

URL

http://hdl.handle.net/10232/6479

PROTEASE ACTIVITY IN PLANT TISSUES (V)

著者

UCHIKOBA Tetsuya, AMAKATSU Kousuke, KANEDA

Makoto

journal or

publication title

鹿児島大学理学部紀要. 数学・物理学・化学

volume

23

page range

139-145

別言語のタイトル

種々の植物組織のプロテアーゼ活性について (v)

URL

http://hdl.handle.net/10232/00004001

PROTEASE ACTIVITY IN PLANT TISSUES (V)

Tetsuya UCHIKOBA*, Kousuke AMAKATSU*, Makoto KANEDA*

Swartz for Leu-PNA, and Hananira, Brodiaea multiflora Benth. for Ala-PNA.

A∫∫ay ofProtect∫｣-Proteolytic activity was measured by two methods.

Caseinolytic activity was assayed by method of Kunitz (#) , with casein as a substrate.

One ml of sample solution was preincubated for 10 min at 30 , and then added to 1 ml of

a solution of 1% (w/w) casein containing 0.02 M phosphate buffer, pH 7.3, at 300,

Af-ter incubation for 30 min the reaction was Af-terminated by the addition of 2 ml of 5%

trichloroacetic acid. After standing for 30 min at room temperature, the precipitate was

removed by filtration through Toyo filter paper No. 5C and the absorbancy at 280 nm of

the trichloroacetic acid-soluble peptides formed was determined with Hitachi

spec-trophotometer 100-60.

The rates of enzymatic hydrolysis of 4-nitroanilide substrates were followed

spec-trophotometrically in 0.1 M Tris-HCI buffer, pH 7.5, at 410 nm with the

A unit of activity was defined as that amount which yielded 0.001 A280nm (0.001

A4iOnm) unit of change per min under the conditions mentioned above. The specific

activity is expressed as the number of enzyme units per 1 ml of Juice.

Results and Discussion

The results of the screening test are shown in Table 1, 2.

Caseinolytic activity was observed in several plants. The activity of the extracts

of bulb of tulip, Tulipa gesneriana L, and Paper mulberry, Broussonetia papyrifera Vent,

As this plant is Moraceae family, it is considered the cysteine protease group of Moraceae

were Chayote, Sechium edule Swartz for Leu-PNA, and Hananira, Brodiaea multiflora Benth.

for Ala-PNA. But these activity units were not enough to study plant aminopeptidase.

Kunitz, M.f (1947) / Gen. Phy∫iol. 30, 291

Whole

Whole

Whole

Whole

Whole

Whole

Whole

Whole

Whole

Whole

Willd.)

Whole

Whole

Whole

Whole

Whole

Whole

Whole

Toogan, White gourd

Whole

Whole

Whole

Tetsuya UCHIKOBA, Kousuke AMAKATSU, Makoto KANEDA*