Abstract
ARPP-16 (cAMP-regulated phosphoprotein of Mr = 16,000) is a substrate for cAMP-dependent protein kinase and is enriched in the basal ganglia. ARPP-16 has been purified to homogeneity from the soluble fraction of bovine caudate nuclei. An additional substrate for cAMP-dependent protein kinase of Mr = 19,000 (ARPP-19) was found to cross-react with rabbit anti-serum prepared against purified ARPP-16. Immunological analysis indicated that ARPP-16 was enriched in the basal ganglia while ARPP-19 was present in similar levels in all brain regions studied and was also present in non-neuronal tissues. ARPP-19 was also purified to homogeneity from bovine caudate nucleus cytosol. Using oligonucleotide probes based on the partial amino acid sequence of purified ARPP-16, cDNA clones for ARPP-16 and ARPP-19 were isolated from a bovine caudate nucleus cDNA library and sequenced. The predicted amino acid sequences of the two proteins were identical except that ARPP-19 had an additional 16 amino acids at the NH2-terminal. The two cDNA clones share an identical 3'-untranslated region of 756 nucleotides. The cDNA clone for ARPP-16 contained 806 additional nucleotides located 3' to this common sequence. The 5'-untranslated regions of the two clones were entirely different. The results suggest the possibility that ARPP-16 and ARPP-19 are produced by alternative transcription and splicing.
Highlights
16,000) is a substrate for CAMP-dependent protein kinase and is enriched in the basal ganglia
Immunological analysis indicated that ARPP-16 was enriched in the basal ganglia while ARPP-19 was present in similar levels in all brain regions studied and was present in non-neuronal tissues
Using oligonucleotide probes based on the partial amino acid sequence of purified ARPP-16, cDNA
Summary
The catalytic subunit of CAMP-dependent protein kinase was purified from bovine heart as described (Kaczmarek et al, 1980) and was stored at 4 “C in 150 mM KPO, (pH 6.8). To monit.or purification, ARPP-16 was assayed by its ability to accept the y-phosphate from [-y-32P]ATP in a reaction catalyzed by the purified catalytic subunit of CAMP-dependent protein kinase, essentially as described for DARPP-32 by Hemmings et al (1984). Phosphopeptides were separated by electrophoresis at 400 V for SO min in the first dimension followed by chromatography in the second dimension in a buffer containing pyridine, l-butanol, water, acetic acid (15:10:12:3). Step 4: HPLC Using Reverse Phase Cls Chromatography-The pooled FPLC fractions were injected onto a Vydac Cie reverse phase column (0.4 x 25 cm, 5-n particle size, 300-A pore size) equilibrated in 0.1% trifluoroacetic acid (v/v). Polyclonal antibodies were raised in two female New Zealand white rabbits by injecting purified ARPP-16 (0.14 mg) conjugated to bovine thyroglobulin with glutaraldehyde and mixed with Freund’s complete adjuvant, as described by Hemmings and Greengard (1986)
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