Planta (Berl.) 130, 141 - 144 (1976)
~ by Springer-Verlag 1976
Iso-Enzymes of Acid Ribonuclease in Cotyledons of Pisum sativum L.. John A. Bryant and Sally C. Greenway, Department of Botany, University College, Cardiff CF1 1XL, U.K. Gillian A. West Nottingham University School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, U.K.
Summary. Acid ribonuclease from cotyledons of P i s u m sativum is very stable, with a temperature opti-
m u m of 65~ It has a molecular weight of 17,500 and there is evidence that a fragment, with a molecular weight of 3,100, retains enzyme activity. Acid ribonuclease from the cotyledons of five-day old seedlings m a y be fractionated into two iso-enzymes, I and II, by CM-cellulose chromatography. The increase in activity of iso-enzyme I is not inhibited by cycloheximide, whereas the increase in iso-enzyme II activity is strongly inhibited by cycloheximide. Cotyledons of 9-day old seedlings contain only iso-enzyme I, whilst cotyledons of 15-day old seedlings contain three iso-enzymes, I, I I a and III.
Introduction In the preceding paper (Bryant etal., 1976), we demonstrated that the development of acid ribonuclease activity in the cotyledons of germinating peas exhibits a biphasic pattern. Preliminary evidence was also presented to show that the early increase in acid ribonuclease is partly independent of protein synthesis. In this paper we report a partial characterisation of acid ribonuclease, including an estimation of its molecular weight. The existence of at least three iso-enzymes is demonstrated, and data are presented which suggest that increases in the activity of one iso-enzyme are not mediated by protein synthesis.
Materials and Methods Growth of Plants was as described in the previous paper (Bryant
et al., 1976). Ribonuctease Assays were carried out essentiaIIyaccording to Sri-
Estimation of Molecular Weight. Cotyledons were homogenised in 0.2 M acetate buffer, pH 5.4, with a pestle and mortar at 2~ The homogenate was centrifuged at 34,000 • for 20 rain at 4~ Ribonuclease was precipitated from the supernatant with 40 to 60% saturated (NH4)2SO 4. The precipitate was dissolved in 0.2 M acetate buffer. The solution was applied to a column of Sephadex G-50, fine (450 x 25 ram) previouslyequilibrated with 0.2 M acetate buffer. Protein was eluted from the column with 0.2 M acetate buffer. Fractions were collected automatically and assayed for absorbance at 280 nm and for ribonuclease activity. Dextran blue, myoglobin, pancreatic ribonuclease, cytochrome C and insulin were used as markers. Partial Purification. The precipitate obtained with 40~50% saturated (NH4)zSO 4 (see above) was dissolved in 0.01 M acetate buffer. Residual (NH~)zSO4 was removed by passage of the extract through a column of Sephadex G-50 (25 x 25 ram). The desalted enzyme solution was applied to a column of carboxy-methyl-cellulose (CM-cellulose) (120 x9 mm), equilibrated at pH 5.4. Protein was eluted from the column first with 0.01 M acetate buffer and then with a linear gradient of 0.0 to 1.0 M NaCI in 0.01 M acetate buffer. Fractions were collected automatically and assayed as described above.
Results and Discussion Stability. Stability of the enzyme was investigated by
storage of crude extracts at I ~ (Fig. 1). At I ~ the enzyme is stable for 4 days. It is also stable for 24 h at 37~ and for at least 24 h at 22~ The temperature optimum, as determined in crude extracts, is 65~ (Fig. 2). The increased activity of the enzyme at high temperature is not caused by changes in the secondary structure of the R N A used as a substrate, since preheating the R N A , followed by rapid cooling to 37~ for assay, does not lead to enhanced activity (Fig. 2). The temperature o p t i m u m is higher than that reported for soluble acid ribonuclease from wheat (Torti et al., 1973), but is similar to that reported for the ribonuclease from bovine pancreas (Davidson, 1972), In fact the general stability of the acid ribonuclease in pea cotyledons closely resembles that o f pan-
J.A. Bryant et al. : Iso-Enzymes of Acid Ribonuclease
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Fig. 1. Effect of storage at I~ ( e s - e ) and dialysis at I~ ( o - o - o ) on the activity of acid ribonuclease in crude extracts from cotyledons of 5-day old seedlings
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Fig. 3. Fractionation of acid ribonuclease on columns of Sephadex G-50 d.
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40 Temperat ure,~
Fig. 2a and b. Effects of temperature on the activity of acid ribonuclease in crude extracts from cotyledons of 5-day old seedlings. (a) Effect of assaying the enzyme at different temperatures. (b) Effect of pre-incubating the substrate for 30 rain at different temperatures, followed immediately by assay at 37~
creatic ribonuclease. The acid ribonucleases from pea leaves (Frisch-Niggermayer and Reddi, 1957) and from cucumber seedlings (Kado, 1968) also show a high level of stability.
Molecular Weight. The enzyme elutes from columns of Sephadex G-50 (fine) as two peaks, A and B (Fig. 3). The molecular weight of the early-eluting peak (A), computed by comparison with the markers, is 17,500+ 1,560. This is within the range shown by acid ribonucleases in other plants: corn, 23,000 (Wilson, 1968), garlic, 20,000 (Carlson and Frick, 1964), cucumber 12,000 (Kado, 1968), potato, 10,000 (Pitt, 1975) and wheat, 9,000 (Torti et al., 1973). The alkaline ribonuclease from pea cotyledons is slightly larger, with a molecular weight of ca. 21,000 (J.A. Bryant and G.A. West, unpublished data). The molecular weight of the late-eluting peak (B) is 3,100 _+530.
This molecular weight is lower than that of any other known enzyme, and it is thus unlikely that peak B represents a native enzyme. It is more probable that this low-molecular-weight ribonuclease is a fragment derived from the native enzyme by proteolysis during extraction. Support for this suggestion comes from the finding that peak B is variable in amount, making up between five and 50% of the total activity. Further confirmation of the existence of a low-molecularweight form of acid ribonuclease comes from results of dialysis experiments. When crude extracts of acid ribonuclease are dialysed at I~ activity is lost (Fig. 1). This contrasts with the maintenance of activity during normal storage at I~ (Fig. 1). The amount of activity lost during dialysis is again variable, the upper limit being 50% of the total activity. It is suggested that this loss of activity is best explained as movement of a low-molecular-weight fragment through the dialysis membrane. A small acid ribonuclease (molecular weight 5,000) has been detected by Pitt (1975) in leaves of potato (Solanum tuberosum) Pitt regards this form of acid ribonuclease as a native enzyme of exceptionally low molecular weight, although the possibility that it is a fragment of a larger protein is not excluded. If these low-molecularweight forms are indeed fragments of the native enzyme, it suggests that acid ribonuclease from certain plants retains enzyme activity even after loss of a large number of its amino acid residues. This suggestion is supported by the finding that acid ribonuclease from pea cotyledons is not inactivated by photodynamic modification of a number of its amino acids (J.A. Bryant and P.S. Phillips, manuscript in preparation).
J.A, Bryant et al. : Iso-Enzymes of Acid Ribonuclease
Iso-Enzymes of Acid Ribonuclease. The chromatographic behaviour on CM-cellulose of acid ribonuclease from cotyledons of five day old seedlings is shown in Figure 4a. Two peaks of acid ribonuclease are present, one eluting in the loading buffer and the other eluting in the NaC1 gradient. The elution of one peak in the loading buffer is not caused b y overloading the column, since the same elution profile is observed when very much smaller quantities of protein are loaded. Further, the two peaks do not represent the high- and low-molecular-weight forms described above, since extensive dialysis to remove the low-molecular-weight form does not alter the pattern of elution from CM-cellulose. These results thus indicate that there are two iso-enzymes (I and II) of acid ribonuclease in the cotyledons of five-day old seedlings. Peas germinated in the presence of cycloheximide at 1 gg/ml do not contain iso-enzyme II but contain the normal amount of iso-enzyme I (Fig. 4b). Since cycloheximide at this concentration severely inhibits protein synthesis (Bryant etal., 1976), these results suggest that the eight-fold increase in acid ribonuclease activity which occurs during the first five days of germination (Bryant et al., 1976) is brought about by two different mechanisms. The increase in the activity of iso-enzyme I is mediated by a post-translational control mechanism, whereas the increase in activity of iso-enzyme II depends on protein synthesis. These results thus extend and confirm the observations presented in the previous paper (Bryant et al., 1976), namely that the development of acid ribonuclease activity in the cotyledons, during the first five days of germination, is only partially inhibited by cycloheximide. Ribonucleases extracted from cotyledons of nineday old and fifteen-day old seedlings (grown without cycloheximide) have also been fractionated by CMcellulose chromatography (Fig. 4c and d). Cotyledons of nine-day old seedlings contain only one isoenzyme, similar in chromatographic behaviour to isoenzyme I. Thus, the decline in acid ribonuclease activity which occurs between day 5 and day 9 (Bryant et al., 1976), is brought about by the disappearance ofiso-enzyme II. The later increase in acid ribonuclease activity (day nine to day fifteen) is accompanied by the appearance of two more iso-enzymes, II a (similar in chromatographic behaviour to iso-enzyme II) and III. The overall patterns of change in acid ribonuclease activity reported in the previous paper (Bryant et al., 1976) are therefore caused by changes in the activities of three or possibly four different iso-enzymes. Iso-enzymes of acid ribonuclease, separable by ion-exchange chromatography, have also been extracted from leaves of potato (Pitt, 1975). Further, the wound-induced increase in acid ribonuclease ac-
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Fig. 4 a-d. Fractionation of acid ribonuclease by ion-exchange chromatography on columns of CM-cellulose. (a) Acid ribonuclease from cotyledons of 5-day old seedlings. (b) From cotyledons of 5-day old seedlings germinated in the presence of cycloheximide (1 gg/ml) (c) From cotyledons of 9-day old seedlings (cl) From cotyledons of 15-day old seedlings
tivity in potato leaves is also brought about by two different mechanisms (Pitt, 1975). The major part of the increase in activity is mediated by a post-translational mechanism, involving an iso-enzyme located predominantly in the lysosomes. The increase in the
activity of the minor iso-enzyme, which makes up ca. 25% of the total, and which is located in the cytoplasm, depends on protein synthesis. The control of acid ribonuclease activity in pea cotyledons is thus remarkably similar to that in potato leaves. The regulation of acid ribonuclease activity at two different levels in cotyledons of Pisurn sativum contrasts with results reported for other germinating seeds. In P. arvense, the early rise in acid ribonuclease activity has been conclusively shown to be completely independent of protein synthesis (Barker et al., 1974), whereas the development of acid ribonuclease activity in germinating barley grains is totally dependent on protein synthesis and is probably mediated by de novo synthesis of enzyme protein (Bennett and Chrispeels, 1972). The data presented in this paper raise a number of points which require further investigation. The mechanism operating to control the activity of isoenzyme I at the post-translational level is unknown; we have as yet been unable to identify any inhibitors of acid ribonuclease in the cotyledons of ungerminated seeds, or any activators of acid ribonuclease in the cotyledons of five-day old seedlings. The location of the iso-enzymes is unknown, although by comparison with potato leaves (Pitt, 1975) iso-enzyme I may be expected to be lysosomal. The properties of the individual iso-enzymes are not known, but comparative structural studies and investigations of substrate specificities are under way in this laboratory.
J.A. Bryant et al. : Iso-Enzymes of Acid Ribonuclease
References Barker, G.R., Bray, C.M., Walter, T.J. : The development of ribonuclease and acid phosphatase during germination of Pisum arvense. Biochem. J. 142, 211-219 (1974) Bennett, P.A., Chrispeels, M.J. : De n o v o synthesis of ribonuclease and/3-1, 3-glucanase by aleurone cells of barley. Plant Physiol. 49, 445447 (1972) Bryant, J.A., Greenway, S.C., West, G.A.: Development of nuclease activity in cotyledons of Pisum sativum L. Planta (Berl.) 130, 137-140 (1976) Carlsson, K., Frick, G. : Partial purification of nucleases from germinating garlic. Biochim. Biophys. Acta 81, 301 310 (1964) Davidson, J.N. : The biochemistry of the nucleic acids. (7. Edt.) London: Methuen and Chapman and Hall 1972 Frisch-Niggermeyer, W., Reddi, K.K.: Studies on ribonuclease in pea leaves. I. Purification and properties. Biochim. Biophys. Acta 26, 40-46 (1957) Kado, C.I. : Purification and properties of ribonnclease isolated from etiolated cucumber seedlings. Archives Biochem. Biophys. 125, 86-93 (1968) Pitt, D. : Changes in activity of lysosomal ribonuclease following mechanical damage to leaves of Solanuum tuberosum L. Planta (Berl.) 123, 125-136 (1975) Srivastava, B.I.S. : Increase in chromatin-associated nuclease activity of excised barley leaves during senescence and its suppression by kinetin. Biochem. Biophys. Res. Commun. 32, 533 538 (1968) Torti, G., Mapelli, S., Soave, C.: Acid ribonuclease from wheat germ: purification, properties and specificity. Biochim. Biophys. Acta 324, 254~266 (1973) Wilson, C.M. : Plant nucleases, II. Properties of corn ribonucleases I and II and corn nuclease I. Plant Physiol. 43, 1339 1346 (1968)
Received 15 December 1975; accepted 14 January 1976