Juno 1991
$l!%3 09ml valwrl~ all, numlw 1. 31$-7iff 6 twtll ~ede~~~t~n rt(l ~nr~~N~ ~j~e~rnl~~ ~~jeYt#~~~~~~~~~i~~~~~~
M~l~~~l~r
cltxiin
and ~~~~~~~~ ~~~l~~i~~~~~~~
enwdin
~~~~-~~~~~t~r~i~ SP_aeductaseof rat liver ~~~~j~~~~ (ISniM q ~it~~~i~~ Noxhirc+, Truiekirs~ Shimoa8~ts’and Kyuiekim I Bqwrlwewl ef#twltM ~~~~~~ii*~~~~~t~~ Sttr~rrty tf tind t Lkptttlmt of Wbchctwlstq, ~ilrudtkut Chkrxlty
Okudaa Wtual
f$&tiriWy,
/b%*x/tlnttr 7-MY~pujr
Reecived4 Mweh I99
I
&nc cneading &;ac.9-kctlaWrui8 S&fedurrw~ wit%Irrilrtttrd frarn rat liver clb)MA librrtrl&$ uning xmtibedier ~p~ifie lirr the enxymr tend aiipnnclautidor 4%prebc’r. fho eDNA tsntaincd TTIII-bar* p;lir opn r&tding Yrttmc~~din&t 317 amino ticid rr~id~x (M, 37 376) und tin unu%uttlly Ian&~Y~unfr#ndttt@d rsgich rich in AT xequclncs in the tatal lengh of 31119b~rc prim. The predicted amino nei& ~qucnee cnvmtinu the #qucnear xlmilitr tu the putntiu+?NADPH- end .W?rtWkintling rcgian& A &NA
d+-24&t~W&l
[email protected];rnc;sBNA elslninfi
ThecDNA
obtaincei was used nf e probe 10 ixalntc a complete cBNA fram there libraries [6], Peairive~y reacted clones isolated tt\railphxcvcral rounds of ircrecnin~ Were subekmcd into p~b%ript
1, INl%QBUCTION
elmc
In bile acids synthesis and steroid hormone metwbolism, d’+ketosteroid S&reductase plays an important role to catalyze reduction of the d”-double bond to give A/B-& conformation [I]. The enzyme W&S recently purified to homogeneity in this laboratory [2,3], Subsequent studies have shown that the Nterminal amino acid of this enzyme is blocked, prompting us to determine the amino acid sequences of peptides obtained by peptidase-treatment and prepare the specific monoclonal and polyclonal antibodies against the enzyme (Onishi et al., to be published). In this paper, WCdescribe the isolation of :I cDNA clone encoding d4-3.kctosteroid S,%rcductase from rat liver cDNA libraries using the specific antibodies and synthetic oligonucleotides corresponding to the partial amino acid sequences as probes. 2. MATERIALS AND METHODS 43-3-Ketostcroid S@reductase was purified from male rat liver cytosol as described previously [2]. Specific polyclonal antibodies were prepared by immunizing BALB/c female mice with the purified protein mixed with Ribi adjuvant as described previously [4]. Oligonucleotides were synthesized based on the amino acid sequence of a peptide fragment of the enzyme ((Lys)-ThrPhe-Ile-AlaNVal-Lys) as follows 5’-TT IAC IGC G(A/T)AT A(G)AA IGT T(C)TT-3’. Tile cDNA libraries were prepared from liver ~oI~(A)~RNA of male rats [S], using Agt 11 and UAP vectors. A Agt 11 oligo(dT)primed cDNA library was screened with the specific polyclonal antibodies and a 32P-labeled family of twelve 20.mer oligonucleotides. Correspondence address: I
$l!%3 09ml valwrl~ all, numlw 1. 31$-7iff 6 twtll ~ede~~~t~n rt(l ~nr~~N~ ~j~e~rnl~~ ~~jeYt#~~~~~~~~~i~~~~~~
M~l~~~l~r
cltxiin
and ~~~~~~~~ ~~~l~~i~~~~~~~
enwdin
~~~~-~~~~~t~r~i~ SP_aeductaseof rat liver ~~~~j~~~~ (ISniM q ~it~~~i~~ Noxhirc+, Truiekirs~ Shimoa8~ts’and Kyuiekim I Bqwrlwewl ef#twltM ~~~~~~ii*~~~~~t~~ Sttr~rrty tf tind t Lkptttlmt of Wbchctwlstq, ~ilrudtkut Chkrxlty
Okudaa Wtual
f$&tiriWy,
/b%*x/tlnttr 7-MY~pujr
Reecived4 Mweh I99
I
&nc cneading &;ac.9-kctlaWrui8 S&fedurrw~ wit%Irrilrtttrd frarn rat liver clb)MA librrtrl&$ uning xmtibedier ~p~ifie lirr the enxymr tend aiipnnclautidor 4%prebc’r. fho eDNA tsntaincd TTIII-bar* p;lir opn r&tding Yrttmc~~din&t 317 amino ticid rr~id~x (M, 37 376) und tin unu%uttlly Ian&~Y~unfr#ndttt@d rsgich rich in AT xequclncs in the tatal lengh of 31119b~rc prim. The predicted amino nei& ~qucnee cnvmtinu the #qucnear xlmilitr tu the putntiu+?NADPH- end .W?rtWkintling rcgian& A &NA
d+-24&t~W&l
[email protected];rnc;sBNA elslninfi
ThecDNA
obtaincei was used nf e probe 10 ixalntc a complete cBNA fram there libraries [6], Peairive~y reacted clones isolated tt\railphxcvcral rounds of ircrecnin~ Were subekmcd into p~b%ript
1, INl%QBUCTION
elmc
In bile acids synthesis and steroid hormone metwbolism, d’+ketosteroid S&reductase plays an important role to catalyze reduction of the d”-double bond to give A/B-& conformation [I]. The enzyme W&S recently purified to homogeneity in this laboratory [2,3], Subsequent studies have shown that the Nterminal amino acid of this enzyme is blocked, prompting us to determine the amino acid sequences of peptides obtained by peptidase-treatment and prepare the specific monoclonal and polyclonal antibodies against the enzyme (Onishi et al., to be published). In this paper, WCdescribe the isolation of :I cDNA clone encoding d4-3.kctosteroid S,%rcductase from rat liver cDNA libraries using the specific antibodies and synthetic oligonucleotides corresponding to the partial amino acid sequences as probes. 2. MATERIALS AND METHODS 43-3-Ketostcroid S@reductase was purified from male rat liver cytosol as described previously [2]. Specific polyclonal antibodies were prepared by immunizing BALB/c female mice with the purified protein mixed with Ribi adjuvant as described previously [4]. Oligonucleotides were synthesized based on the amino acid sequence of a peptide fragment of the enzyme ((Lys)-ThrPhe-Ile-AlaNVal-Lys) as follows 5’-TT IAC IGC G(A/T)AT A(G)AA IGT T(C)TT-3’. Tile cDNA libraries were prepared from liver ~oI~(A)~RNA of male rats [S], using Agt 11 and UAP vectors. A Agt 11 oligo(dT)primed cDNA library was screened with the specific polyclonal antibodies and a 32P-labeled family of twelve 20.mer oligonucleotides. Correspondence address: I
