Protease activity from maize pollen

Radłowski, Marek; Kalinowski, Andrzei; Krolikowski, Z; Bartkowiak, S.

doi:10.1016/s0031-9422(00)90625-3

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…!n all purification steps, the fractions with proteolylic activity were collected, dialysed against ati appropriate buffer and concentrated. The results are presented in Tab, 1 and Fig, I, After five steps, we reached the same degree of purification (46-fold) as in the case of serine protease (,i2-fold) from the same source (Radlowski et al, 1994a Durittg our studies oti diffusible proteins front tnaize pollen, we found that some had proteolytic activity. The prelitninary experiments indicated different properties of the eluted enzytne than those of the serine protease from the same source.…”

Section: Resultssupporting

confidence: 58%

“…In contact with the sttgma surface, the pollen grain secretes a droplet of liquid containing the enzytnes that hydrolyze external stigmatic substrates (Stanley and Linskens 1985), It has long been thought that protein diffuses passively from the pollen, but experiments with metabolic poisons suggest that tt must be partially an energy-driven process (Jackson and Kamboj 1986), The activity of hydrolases such as amylase, pectinase and cutinase in pollen secretions has been considered (Stanley and Linskens 1985), We have nO' information about the release of proteolytic enzymes, but they have not been extensively sttidied in pollen (Brewbaker 1971), Furthermore, the presence of proteases in the pollen wall was confirmed by fluorescence and substrate-film methods (Bhalla et al, 1986, Knox 1971, Previously, we reported on the isolation, purification and some properfies of a protease from maize pollen (Radlowski et al, 1994a) and suggested a possible role during pollen germination (Radlowski et al, 1994b), In this paper we report on the activity of another pro-teolytic enzyme located in the pollen wall of the same material, using silk proteins as substrates. The properties of both enzymes are compared and their possible influence on pollen tube growth considered.…”

Section: Introductionmentioning

confidence: 99%

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Proteolytic activity in the maize pollen wall

Radłowski¹,

Kalinowski²,

Adamczyk³

et al. 1996

Physiol Plant

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S. 1996. Proteolylic activity in the maize pollen wali, -Physiol. Plant, 98: i72-17S, A new protease from maize {Zea may.'; L,) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid ehromatography, SDS-PAGE and HPLC showed that the enzyme ha,s a dimeric stracture of M, ca 60 000, Inhibitor investigations indicated an aspartic acid residue in it,s active site. The optimum pH for maize pollen aspartic proteinase activity was 5,6, and the optimum temperature was 45°C, The enzyme is easily eluted from the pollen grains and, as confimied by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn'*, The pi value for purified aspartic proteinase, as estimated after IEF, was 5,0, Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tuix germination. The properties of serine and aspartic proteinases from maize pollen are compared.

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Section: Resultssupporting

confidence: 58%

Section: Introductionmentioning

confidence: 99%

Proteolytic activity in the maize pollen wall

Radłowski¹,

Kalinowski²,

Adamczyk³

et al. 1996

Physiol Plant

Self Cite

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“…Proteases from a limited number of allergenic pollens have been purified (e.g. [ 17 - 19 ]), but to date there is only one set of reports suggesting that a pollen allergen might have protease activity [ 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

et al. 2003

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BackgroundPollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation.MethodsPollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates.ResultsAll pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr ~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1.ConclusionOne- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1.

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Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants ofLolium multiflorum Lam.

2006

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Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas of Lolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

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Protease activity from maize pollen

Cited by 7 publications

References 27 publications

Proteolytic activity in the maize pollen wall

Proteolytic activity in the maize pollen wall

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants ofLolium multiflorum Lam.

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