Partial Purification and Characterization of a Phytochrome-degrading Neutral Protease from Etiolated Oat Shoots

Pike, Carl S.; Briggs, Winslow R.

doi:10.1104/pp.49.4.521

Cited by 53 publications

(31 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ten mm 2-mercaptoethanol and L-cysteine each enhanced its activity (Table I). Thus bean protease was found to belong to the so-called sulfhydryl-dependent proteases which is common in plant tissue (as reported, for example, by Pike and Briggs in Avena [6]). …”

Section: Resultsmentioning

confidence: 94%

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L.

Yomo¹,

Srinivasan²

1973

Plant Physiol.

View full text Add to dashboard Cite

In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The Rapid liberation of low molecular weight nitrogenous compounds at the expense of stored proteins at the early stage of bean germination was reported by Pusztai and Duncan (7). They also reported in that paper a time course of protease activity which reached maximum on the 8th day of germination. Seeschaaf and Pirson (8) reported that in half cotyledons of blue lupine, protein breakdown proceeded, and the amounts of protease and several other enzymes, including 6-phosphogluconic acid dehydrogenase, increased during incubation of detached cotyledons more than those in attached cotyledons did during germination. They found that the increase occurred only in the absence of the buds; removal of roots or hypocotyl had no effect. Addition of IAA to incubated cotyledons inhibited the increase of 6-phosphogluconic acid dehydrogenase activity. They concluded that auxin from the bud controls enzyme levels in the cotyledons. However, there are no data to show whether auxin has a similar effect on digestive enzymes. a-Amylase activity was also reported to increase dur1Brooklyn Botanic Garden Contribution No. 203. This work was supported by Grant GB 32196 from the National Science Foundation.ing both germination and incubation of axis-free half cotyledons of beans by Dale (1) and by Gepstain and Ilan (2). Dale suggested that gibbereilin is involved in a-amylase formation, while Gepstain and Ilan observed an enhancing effect of kinetin. Yomo has reported that formation of a-amylase in detached bean cotyledons was inhibited by ABA during incubation (10). Yomo and Varner (11) found that in detached cotyledons (a) a-amylase activity was also inhibited by ABA, and (b) protein breakdown and protease formation were very slight compared with attached cotyledons. We report here on the protein breakdown and the formation of protease in comparison with that of a-amylase, during incubation of excised bean cotyledons, in relation to effects of cycloheximide and ABA. MATERIALS AND METHODSGeneral Procedure. Bean (Phaseolus vulgaris L. cv. Kentucky Wonder) seeds were sterilized in 1 % aqueous NaOCl (Allied Chemical) solution for 10 min and imbibed at 22 C for 16 hr. Imbibed seeds were germinated on moist vermiculite in the dark. Cotyledons from germinated seedlings are referred to as attached cotyledons. For incubation experiments, the imbibed seeds were cut transversely in half and the axisfree half cotyledons (detached cotyledons) were sterilized for 1 min in 0.2% NaOCl solution which had been neutralized with 2 N HCI just before use. They were then put in a 125-ml Erlenmeyer flask containing 10 ml of 2% agar solution whic...

show abstract

Section: Resultsmentioning

confidence: 94%

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L.

Yomo¹,

Srinivasan²

1973

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…Phytochrome from many dicots shows reversion and destruction in vivo (17, 18), though there are at lease two plants, Amnaranthus (19) and pumpkin (2), which show only Pfr destruction. Partially purified extracts often show mainly reversion (10, 27), especially at temperatures near 0 C, though there is a recent report of in vitro destruction (24).Work reported elsewhere (29,30) rye phytochrome was also degraded by the protease, yet the phytochrome alone was quite unstable to incubation at 25 C, so evidently other factors are involved in loss of photoreversibility in vitro. With a knowledge of the properties of the protease (29, 30), experiments reported here were undertaken for assessment of the involvement of the enzyme in in vivo destruction by the use of suitable inhibitors and activators.…”

mentioning

confidence: 98%

“…Work reported elsewhere (29,30) sought to explain the instability of crude extracts of oat phytochrome. A neutral proteolytic enzyme was found to be present in such preparations and to be at least partly responsible for this loss of photoreversibility in vitro.…”

mentioning

confidence: 99%

“…Highly purified (and protease-free [33]) 1 Research was supported by grants from the National Science Foundation (GB-15572) and E. I. duPont de Nemours and Co. rye phytochrome was also degraded by the protease, yet the phytochrome alone was quite unstable to incubation at 25 C, so evidently other factors are involved in loss of photoreversibility in vitro. With a knowledge of the properties of the protease (29,30), experiments reported here were undertaken for assessment of the involvement of the enzyme in in vivo destruction by the use of suitable inhibitors and activators.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Dark Reactions of Rye Phytochrome in Vivo and in Vitro

Pike¹,

Briggs²

1972

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo.The effects of various inhibitors of an in vitro phytochromedegrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 nmolecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.Though the destruction and reversion reactions of phytochrome have long been known in vivo (7,17), their precise role in the physiological action of the pigment system has not been established. Reversion had been assigned a major role in photoperiodic timing (3, 16), but more recent work has questioned this view (12). Many of the "paradoxes" of phytochrome physiology involve these reactions (17). For example, though reversion can easily be detected in partially purified phytochrome from various grasses (8,10,27), no in vivo reversion of coleoptile tip phytochrome has ever been seen (6, 14. 31). Phytochrome from many dicots shows reversion and destruction in vivo (17, 18), though there are at lease two plants, Amnaranthus (19) and pumpkin (2), which show only Pfr destruction. Partially purified extracts often show mainly reversion (10, 27), especially at temperatures near 0 C, though there is a recent report of in vitro destruction (24).Work reported elsewhere (29,30) rye phytochrome was also degraded by the protease, yet the phytochrome alone was quite unstable to incubation at 25 C, so evidently other factors are involved in loss of photoreversibility in vitro. With a knowledge of the properties of the protease (29, 30), experiments reported here were undertaken for assessment of the involvement of the enzyme in in vivo destruction by the use of suitable inhibitors and activators.Recent work in our laboratory (15) has shown that the previously studied 60,000 mol wt phytochrome unit (27, 33) is a stable fragment produced by limited proteolysis. Indeed, a much larger molecule has now been isolated and studied. We feel that this material (about 9 S) is probably native phytochrome. Its absorption spectrum is identical (in the region above 500 nm) to that of the fragment.The previous work on in vitro reversion of highly purified grass phytochrome can be summa...

show abstract

“…Thus, the Killerphytochrome interaction may lead to the development of a new, much needed assay for phytochrome in vitro, other than difference spectrophotometry. Recent observations (12,17) suggest that crude oat phytochrome preparations contain an endoprotease which converts the native phytochrome into a smaller 60,000 mol wt species.…”

Section: Methodsmentioning

confidence: 99%

Loss of Phytochrome Photoreversibility in Vitro

1975

Plant Physiol.

View full text Add to dashboard Cite

Partial Purification and Characterization of a Phytochrome-degrading Neutral Protease from Etiolated Oat Shoots

Cited by 53 publications

References 49 publications

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L.

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L.

The Dark Reactions of Rye Phytochrome in Vivo and in Vitro

Loss of Phytochrome Photoreversibility in Vitro

Contact Info

Product

Resources

About