purified Tennessee Winter and Dicktoo enzymes.
Dinucleoside Cleavage R ate
(nmole/minZlO units of RNase) lsd1 monophosphate Tenn. Winter Dicktoo Dicktoo
3'---+ 5 ' P-lOOA P-lOOA1 P-lOOA2 (0. 05)
GpA* 1. 04 0. 95 0. 82 0. 04
GpC 2. 09 1. 19 1. 55 2. 51
GpU 7 - 85 7. 79 7. 36 3. 92
UpG* * 3. 04 2. 18 2. 00 0. 34
UpC 0. 46 0. 41 o·. 67 0. 26
UpU 0. 99 0. 69 1. 09 0. 68
ApA 0. 58 0. 39 0. 41 0. 36
ApG 3.17 2. 60 3- 39 1. 02
ApC* * 6. 57 ·0. 89 0. 88 0. 86
ApU* * 7. 71 1. 18 1. 22 0. 77
CpA 1. 52 1.18 1. 32 0. 59
CpG* * 4. 27 1. 43 1. 66 1.15
CpC* * 1. 42 0. 73 0. 67 0. 36
CpU* * 1. 13 0. 34 0. 40 0. 58
1 Least significant difference at 5% level.
* Significantly different among all three enzymes.
** Significantly different between the Tennessee Winter enzyme and Dicktoo P-lOOA1 or P-lOOA2•
64
significantly.different for all three enzymes. ApC, ApU, CpG, CpC and CpU were each cleaved by purified Tennessee Winter enzyme at a significantly faster rate than by either of the purified Dicktoo enzymes. The only signif
icant difference between the two Dicktoo enzymes wa� the rate of cleavage of GpA which might require further
substantiation.
The dinucleoside monophosphates are grouped according to the order of rate of cleavage by these three enzyme preparations in Table IV. The GpU substrate is seen to be cleaved most rapidly by all enzymes . ApA and UpC were
(P-lOOA), while UpC, ApA and CpU were cleaved most slowly by the _purified Dickto_o P-lOOA1 and P-lOOA2 enzymes. ApU, ApC and CpG were in the group with the highest .cleavage rate by purified Tennessee Winter enzyme, but this was not the case for the two purified Dicktoo enzymes . The rates of hydrolysis of these three dinucleoside monophosphates were also found to be significantly different, when
comparing the Tennessee Winter enzJ7Jile with the two Dicktoo enzymes .
The major difference between the Tennessee Winter P-lOOA enzyme and both Dicktoo P-lOOA1 and �2 was the cleavage rate of the diester linkage between an adenosine
pyrimid�e dinucleoside monophosphate (ApX) . The Tennessee
65 Table IV. Order of cleavage rate of dinucleoside mono
phosphates by the purified Tennessee Winter and Dicktoo enzymes.
1 Tenn. Winter Dicktoo Dicktoo .R ange of cleavage rate P-lOOA P-lOOA1 P-lOOA2
1 . 8 to 4. 5 GpU Gp1J GpU
ApU ApC CpG
2. 3 - 5 to 2. 5 .A.pG ApG · ApG
UpG UpG UpG
GpC
3 - 1. 5 to 0. 6 CpA CpG G:pG
CpC GpC GpC
CpU CpA CpA
GpA ApU .ApU
UpU Gp.A UpU
.ApC .ApC
CpC GpA
UpU CpC
UpC
4. 0. 6 to 0. 3 ApA UpC .ApA
UpC .ApA CpU
CpU 1 nrnole/min/10 units of RNase.
66
Winter enzyme cleaved ApX (X as either one of the two
pyrimidine bases) at a rate seven .times faster than Tiicktoo enzymes (Table III). The two enzymes from Dicktoo were not significantly different in terms of substrate
specificities.
In yeast RNA, the chance of adenine base having a pyrimidine base directly adjacent would be one-half.
Therefore, when yeast RNA was used in the assay, the
Tennessee Winter enzyme would hydrolyze the RNA chain into smaller oligonucleotides, and eventually mononucleotides, at a faster rate than the Dicktoo enzymes.
Tabl 3G 1.T o..nd YI m�....,.T"'8.rizo t�c stq}'::ioc yiclc. of the purification process for RNases from Tennessee Winter and
Dicktoo plants, respectively.
Table V. Purification of Tennessee Winter RNase .
Table VI. Purification of Dicktoo RNase .
69
CO NCLUSIO NS
In the present investigation, the need to define differences in R NA-degrading enzymes between the two phenotypes has resulted in the development of a purifica
tion scheme. Conclusions drawn from the results of these studies are summarized as follows :
1. The purification of R NA-degrading enzymes from barley seedlings was complicated by contaminating enzymes; these contaminants were active upon the substrate, p
nitrophenyl phosphate. A sequence of anion exchange chromatography, preparative electrophoresis and gel fj_lti:.ca ti011 wat> .found -Lo be the most e.ffec;tiv e technique for removal of these contaminating enz�es.
2. The principal groups of RNA-degrading enzymes present in barley seedlings, as shown by electrophoresis, were an EDTA-sensitive group and another group · not affected by this chelating agent. According to properties listed in published reports, the first group are nucleases, whereas the· second group are R Nase I type enzymes. The identity of these enzymes was deduced from electrophoretic mobility , pH optima and response to ionic condition.
3. The rnicleases from both cultivars shoFed suppres
sion of activity by high- ionic conditions ; the degree ·of suppression was about equal in the assays for each cultivar.
70
The higher residual activity previously observed in the crude extract of Tennessee Winter at high ionic conditions was attributed to a greater potential for RN"A degradation by the EDTA-insensitive (RN"ase) fraction from this cultivar in comparison to the Dicktoo cultivar.
4. The RNase preparation obtained by preparative
electrophoresis from -Tennessee Winter extracts, had a�tivity peaks with isoelectric points of
4. 9 , 5 . 8
and a trace of activity at5.3 ,
while a similar R.Nase preparation from Dicktoo had isoelectric points of4 . 9 , 5 . 8
and 6. 1. The nuclease preparation from Tennessee Winter contained one peak with an i soelectric point between 5 - 3 to ·5 . 5 , while the Dicktoo nuclease preparation exhibited three peaks having ·isoelectric points of4.9 , 5 .5
and 6. 0. These tentative findings require further verification using the final purified preparation.5.
The major difference between the purified RNases from Dicktoo and Tennessee Winter was in the cleavage rates of ApC and ApU. Tennessee Winter RNase was able to cleave the phosphodiester bond between adenosine and a pyrimidine at a faste� rate than was Dicktoo RNase.6. These results confirm previous reports that a phenotypic variation exists i� RNA-degrading enzymes of winter barley. The primary distinction between cultivars is in RNase I type enzymes. It is not possible at this
71
time to conclude whether Tennessee Winter (the non-hardy cultivar) contains more of this enzyme than does Dicktoo
(the hardy cultivar), or whether the enzyme from Tennessee Winter is simply more active on the substrates tested.
72
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73
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75
38.
--==---=---
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