ANALYTICAL BIOCHEMISTRY 83, 632-635 (1977)

A Sensitive Radioimmunoassay for lsopentenylacienosinel

SALEEM A. KHAN,~ M.Z. Hu~AYu~,~ AND T. M.JACOB~

Drpurttnerrt of‘ Biociwnlistr~, Imliun ftwtitctte of Science, Bang&m+560012, India

Received May 5, 1977; accepted July 14, 1977

A sensitive radioimmunoassay for the nucleoside isopentenyladenosine (iA) by using iA-specific antibodies and a nitrocellulose membrane filtration technique is described. The reliability of the method is demonstrated by using specific tRNAs of known structures for the estimation of iA in their digests. This assay can be used to quantitate minute amounts of iA in the presence of a large excess of other nucleosides.

One of the reasons for preparing anti-nucleic acid antibodies is the possibility of utilizing such antibodies as specific chemical probes in nucleic acid research. Antibodies to a modified nucleoside can be very useful in detecting and quantitating the modified nucleoside in a given nucleic acid preparation or biological sample. Antibodies to the hyper- modified nucleoside N6-(A2-isopentenyI)-adenosine (iA) have already been raised and characterized (l-4). The antibodies were found to be highly specific for iA (3). iA is known to occur at the 3’ end of the anticodon of certain tRNA species from a wide variety of sources, like mammals, plants, and microorganisms (5,6). In spite of many studies, the role of iA in tRNAs is yet to be established.

In this paper we demonstrate that specific antibodies to iA can be used in a simple, rapid, and sensitive assay for this interesting hypermodi~ed nucleoside in tRNA digests. We hope that the assay described will be useful in a number of fields, since iA, apart from its little known func- tion in tRNA, is a potent cytokinin (4) and is known to have a bewilder- ing variety of biological actions (5,7-10). We have described the prepara- tion and characterization of anti-iA antibodies earlier (3).

MATERIALS AND METHODS

iA was purchased from Sigma Chemical Company. Bacterial alkaline phosphatase and snake venom phosphodiesterase were from Worthington

t This is No. 4 in the series entitled ~‘Immunological studies on nucleic acids and their components”; No. 3 is Ref. (3).

z Present address (to which reprint requests should be sent): Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, New York 10016.

3 Principal investigator. 632

Copyright <’ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved. ISSN 0003-2697

RADIOIMM~NOASSAY FOR ISOPENTENYLADENOSIN~ 633

FIG. 1. Standard curve for the estimation of iA, based on the inhibition of [*H]iA-anti- iA binding. The reaction mixture contained 0.1 ml of 1500 diluted anti-iA serum, 0.1 ml of Tris-HCI-buffered saline [TBS; 0.14 mM NaCI, 0.01 M Tris-HCI (pH 7.9, 5.10m4~ Mg*+, 1.5.10-’ M Ca2+] containing 3 pmol of [3H]iA, and 0.1 ml of nonradioactive iA in TBS. After incubation at 37°C for 10 mm, the contents were filtered on prewetted nitro- cellulose membrane filters, washed with TBS, dried, and counted. Control (no inhibitor) bound 3500 cpm above a normai rabbit serum (~:5O~difuted~ blank, which bound 200 cpm. Each value represents the average of two experiments: the variation between duplicates was less than 2%.

Biochemical Corporation. The preparation and characterization of anti-iA antibodies have been described (2,3). Rat liver tRNA was prepared by the method of Hardesty et at. (I I). Rat liver tRNA,+,ser was a gift from Dr. W. Wehrli. Unfractionated tRNA, tRNASer, and tRNAP”’ from yeast were gifts from Dr. H. G. Zachau. E. coli tRNAZTYr, tRNAphe, tRNA,““, tRNAArg, and tRNAfMet were gifts from Dr. S. Nishimura. For the estimation of iA, an ezfiSrnax value of 20,000 was used. The mem-

TABLE I

ESTIMATION OF iA IN DIFFERENT tRNA DIGESTS”

Source tRNA Amount of tRNA

digest Lug) Percentage inhibition

iA iA 0x1 (%)

Rat liver

Yeast -do- Rat liver E. co/i -do- -do- -do- -do- Yeast

Unfraction- ated tRNA

-do- ,RNAS” tRNA,,,“” tRNAzTy’ tRNAP”’ tRNAA” tRNA,“” tRNA,“” tRNA’h’

25

25 I

30 30

100 100 100 100

56 II.0

53 9.7 0.0388 49 8.7 0.87 45 7.0 0.70 43 6.0 0.02 43 6.0 0.02

0 0 0 0 0 0 0 0 0 0 0 0

0.044

o The pH]iA-anti-iA system was used for inhibition analysis, as described in the legend for Fig. 1.

634 KHAN,HUMAYUN,ANDJACOB

brane binding assay was done as described earlier (12). The preparation of [ 3H]isopentenyladenosine (trialcohol derivative) has been described earlier (2).

tRNA digestion with enzymes. Different tRNA samples were digested to nucleosides by the combined action of snake venom phosphodiesterase and alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.5) for 16 h at 37°C. Nucleoside concentrations were determined by uv absorption (30 Azso units were taken as 1 mg of nucleosides).

RESULTS

Figure 1 shows the results obtained with various quantities of -an authentic iA sample used to inhibit the binding of [3H]iA to anti-iA anti- bodies. There is a linear relationship between the extent of inhibition and the iA concentration for the range used. Table 1 gives the extent of inhibition caused by various tRNA digests on the same system and cor- responding values for iA obtained from Fig. 1. Thus, 25 pg of unfrac- tionated tRNA digests from rat liver and yeast give inhibition values equal to those given by 11 and 9.7 ng of iA, which in turn correspond to an iA concentration of 0.044 and 0.0388%, respectively. These values for iA are close to the values reported by other methods (5).

A purified preparation of yeast tRNA Ser (known to contain iA) gives an inhibition value corresponding to an iA content of 0.87% of the total nucleosides or 0.74 mol of iA/mol of tRNA. A rat liver tRNA,+,s” prepara- tion (theoretically containing 0.7 mol of iA/mol of tRNA) similarly shows an iA content of 0.7% (0.6 mol of iA/mol of tRNA). Both these values are close to the expected values, although slightly low, probably because of either contamination with non-iA containing tRNA or destruction of iA during storage of tRNA.

It is interesting to note that E. co/i tRNAzTY’ and tRNA’h’, in which 2-methylthio-N6-isopentenyladenosine replaces iA, are very poor inhibitors of the [3H]iA-anti-iA binding and give inhibition values corresponding to an iA content of 0.02% of the total nucleosides. tRNAmMet, tRNAArg, and tRNAtMet from E. co/i and tRNA Phe from yeast, which do not contain iA, give no inhibition at the highest concentrations used.

DISCUSSION

The results presented in this paper show that the anti-iA antibodies can be used to quantitate the iA content of tRNAs. Small quantities of various unfractionated tRNA digests (25 pg) were sufficient to quantitate the iA content. Extremely small amounts (1 pg) of purified tRNAs con- taining iA were adequate for estimating the amount of iA present in these tRNAs.

That the inhibition obtained with tRNA digests is specifically due to their iA content is shown by the fact that no inhibition whatsoever is observed

RADIOIMMUNOASSAY FOR ISOPENTENYLADENOSINE 635

with as much as 100 pg of digest of tRNAs known to lack iA. This is confirmed by an earlier study made in this laboratory (MZH, Ph.D. thesis), in which it was shown that a tRNA preparation containing iA could in- hibit the [ 3H]iA-anti-iA system, but not after specific chemical destruc- tion of iA.

It is interesting that E. co/i tRNA.,‘r”’ and tRNAi’h” containing 2-methyl- thio isopentenyladenosine could inhibit the [ 3H]iA-anti-iA binding to only a very small extent, showing that anti-iA antibodies specifically recognize isopentenyladenosine and that even very closely related nucleo- sides are unlikely to cause significant interference. Apart from 2-methyl- thio iA, the purified tRNAs tested contained the following rare nucleosides (13): pseudouridine; 4-thiouridine; 5,6-dihydrouridine: 3-(3-amino-3-car- boxypropylhrridine; N”-acetylcytidine; 2’-U-methylcytidine: 5-methyl- cytidine; 2-thiocytidine: inosine: I-methyladenosine; 2-methyladenosine; Nfi-( N-threonylcarboxyl)-adenosine: Nz-dimethylguanosine; 2’-O-methyl- guanosine: N7-methylguanosine, and nucleoside Y. Obviously, none of these nucleosides interfered with the assay.

The method described in this report can be readily adapted to detect minute quantities of iA in biological fluids. Results presented elsewhere show that this immunochemical technique can be used to quantitate the free iA present in the rice embryo at different stages of its growth (14).

ACKNOWLEDGMENTS

This research was supported in part by Grant FG-In-474 (to TMJ) from the United States Department of Agriculture, Agricultural Research Service, authorized by Public Law 480. SAK acknowledges receipt of a senior research fellowship from CSIR, India.

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