Hum Genet (1990) 84:125-128

9 Springer-Verlag 1990

Molecular cloning of human preproacrosin cDNA Ibrahim M. Adham, Uwe Klemm, Wolf-Martin Maier, and Wolfgang Engel Institut fiir Humangenetik der Universit~it, Gosslerstrasse 12d, D-3400 G6ttingen, Federal Republic of Germany

Summary. Complementary DNA-clones for human preproacrosin have been isolated from a human testis c D N A library in ~,gtll. The nucleotide sequence of the 1402bp c D N A insert includes a 20 bp 5' noncoding region, an open reading frame of 1263bp corresponding to 421 amino acids (45.9 kdalton), and a 105 bp 3' untranslated region. The deduced amino acid sequence is compared with that recently evaluated from a c D N A clone for boar preproacrosin. The sequence identity is 70%; the leader sequence, the catalytic triad (His, Asp, Ser; which is characteristic for serine proteinases) and the positions of the cysteine residues crosslinking the light and the heavy chain of the active enzyme, acrosin, are conserved in both species. A t the C-terminal end, a proline-rich sequence is present in both species; this may represent the species-specificity of acrosin.

Materials and methods Isolation o f c D N A clones

A k g t l l human testis c D N A library (Clontech, Palo Alto, Calif., USA) was screened with a nick-translated boar preproacrosin ~.BA2 cDNA that comprises the nucleotides 652-1051 of boar preproacrosin c D N A (Adham et al. 1989). Hybridization was carried out in 5 • SSPE, 5 x Denhardt's solution, 0.1% SDS, 200 g/ml denaturated salmon sperm D N A and the probe, at 65~ overnight. Filters were washed twice at 60~ to final stringency at 2 • SSPE, 1% SDS. Insert D N A was subcloned into pUC8 and sequenced by the dideoxy chain termination method (Sanger et al. 1977). To resolve compressions in G/C regions of the cDNA, the method of Maxam and Gilbert (1980) was used. Northern blot analysis

Introduction Acrosin (EC 3.4.21.10), a sperm acrosomal serine proteinase has been implicated in the recognition, binding and penetration of the zona pellucida of the ovum (McRorie and Williams 1974; Jones et al. 1988; T6pfer-Petersen and Henschen 1988). The enzyme is stored in the sperm acrosome as a zymogen form, proacrosin, which is activated to the mature enzyme during the so-called acrosome reaction (Polakoski and Parrish 1977). Despite the great importance of acrosin in the fertilization process, very few data regarding its molecular structure are available, and these come mostly form the boar (FockNtizel et al. 1984; Mfiller-Esterl et al. 1984). In this species, the single chain glycoprotein of proacrosin has an estimated molecular mass of 55 kdalton. During its activation, the two chain glycoprotein acrosin is formed with the light chain (4.2 kdalton) and the heavy chain (37 kdalton) being held together by two disulfide bridges. Only the light chain and small regions of the heavy chain, including the histidine site and the serine active site, have been sequenced. Recently, we reported a boar c D N A clone of 1418 bp that codes for the total amino acid sequence of a preproacrosin, 416 residues in length (Adham et al. 1989). The preproacrosin was found to contain a hydrophobic leader sequence of 15 amino acids, and, at the C-terminal end, a domain of 127 amino acids rich in proline residues that has not been found in any other serine proteinase studied to date. This domain may represent the binding site of acrosin to the zona pellucida of the ovum (Adham et al. 1989). Here, we describe the c D N A sequence of human preproacrosin and its expression in the mammalian testis. Offprint requests to: W. Engel

Total R N A from testes of 11 different mammalian species including human was isolated by the guanidinium isothiocyanate method (Chirgwin et al. 1979). Samples of total R N A were denatured in 33% formamide, 6% formaldehyde at 65~ for 10 min. Total R N A (20 pg) was resolved on a 1% agarose gel containing 6% formaldehyde and then transferred to nitrocellulose filters. The R N A blots were hybridized with the 32p-labeled 1.15 kb E c o R I / E c o R I fragment of human preproacrosin c D N A at 65~ under the same conditions as described for testis c D N A library screening. The filters were washed to final stringency in 0.7 x SSPE, 0.1% SDS at 60~ Autoradiography was performed using hyperfilm MP (Amersham).

I

5'

KE

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I

I

S

T

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I I

I

J

3'

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J

~HAIo

~,HA 2

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Fig.1. cDNA clones and sequencing strategy for human preproacrosin. The cDNA clones )~HA2, )~HA6 and )~HAa0 are represented by lines. The solid bar represents the coding region for human preproacrosin, the thin lines indicate untranslated regions. Restriction sites used for subcloning into pUC8 and subsequent sequencing are marked as K: KpnI, E: EcoRI, S: StyI, T: SstI. Arrows indicate the extent and direction of the subcloned strands that were sequenced

126 -20

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60

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GGCCAGGCAG~'GCCAGGAGT'ATG GTT GAG AT(; CTA CCG ACT GCC ATT CTG CTG GTC TTG GCA GTG TCC GTG GTT GeT AAA GAT AAC GCC ACG TG~ Net Val GIo Net [~u Pro ~hr Ala lie L e u L e u V a l ~ e u A l a V a l S e t V a l V a l A l a L y s Asp Asn A l a T h r C y s

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90

80 __-

100

110

120

130

140

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GAT GGC CCC TGT GGG T T A eGG ~ Asp GIy Pro Cys GIy Leu Arg Phe

170

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180

AT~ ~ Net Val

Trp

350

210

Thr

370

1"yr A s h S e t

His

380

540

710

Asn

390

GIy

560

640

c~e c~r ~c

Cys

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Arg Ala

420

430

480 ATT GGG c ~ c Ile GIy Pro

500

490

~c

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cT~ CCC C~c ~

G1y C y s I ~ u

570

Pro

His

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580

510 ~A

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Lys Ala Gly Leo

590

520

c c c AGA G ~

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Arq

600

GIy

670

680

690

730

740

750

760

770

780

790

9

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276

910

920

930

940

950

960

970

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CAA TCG GCC ACC C C T CCA CCT CCC ACC ACT CGA CCG CCC CCG A'l~r CGA CCC CCC ~ ~C G i n S e r A l a ~Phr P r o P r o P r o P r o T h r T h r A r g P r o P r o P r o l i e A r g P r o P r o P h e S e t

990

1000

1010

1020

1030

1040

CAC CCT ATC T C T GCT CAC C'rT CCT His Pro Ile Ser Ala His Leo Pro 1050

1080 ~C &la

1090

1100

1110

1120

1130

1140

1150

9

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TCA C C T ' I ~ A CCC CCA CCC CCA CCC CCA CCC CCA CCT ACA CCC "rCA T C T ACC ACA AAA C I ~ CCC CAA GGA C ' r l ' ' I ~ T ~ S e r P r o L e u P r o P r o P r o P r o P r o P r o P r o P r o P r o T h r P r o S e r S e r T h r T h r L y s Leu P r o G i n G I F L e u S e t Phe

1160

1180

1170

9

306

1060

~ CAA CCG CCC CCT CGA CCA C'I~ CCA CCC CGA CCA CCG GCA GCC CAG CCC CCA CCC CCA CCT ' I ~ A CCC CCG CCC CCA CCC CCA Phe Gln Pro Pro Pro Arg Pro Leo Pro Pro Arg Pro Pro Ala Ala Gin Pro Pro Pro Pro Pro Ser Pro Pro Pro Pro Pro Pro

1070 CCTCCA Pro Pro

216

810 820 830 840 850 860 870 880 : . : : . . A A ~ CGC CCC GGA ATe T&C ACA C~C ACC TGG CCC TAT CTG AAC TGG ATe GCC TCC AAG ATT GGT TCT AAC GCT TTG CGT L y s A r g P r o G I y T i e T y r T h r A l a T h r T r p P r o T y r L e o Asn T r p T i e A l a S e r L y s I l e G l y S e t Ash A l a L e u A r g

900

TAT Tyr

186

700

9

980

156

610

890 A'I~ A'~ Met Ile

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AAA GCC CCC AGG CC& TeA TCT ATA CTG ATG GAG GC& CGT GTG GAT CTC ATe GAC Lys Ala Pro Arg Pro Set Ser Tie Leo Net GIu Ala Arg Val Asp Leu Ile Asp 660

126

246

4k

800

650

410

GGT GGG CCT CTC ATG TGC AAA GAC AGC AAG GAA AGC GCC TAT GTG GTC GTG GGA ATC ACA AGC TGG GGG GTA GGC G l y G I y P r o L e u Met C y s L y s A s p S e r L y s G l u S e r A l a T y r V a l V a l V a l G I y I l e T h r S e r T r p G l y V a l G I y

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470

400

A ~ CAT GAA AAA TAC AAC TCT GCG ACA GAG GGA AAT GAC A T T GCC CTC T i e H i s G I u L y s T y r Ash S e r A l a T h r G l u G I y A s n A s p I l e Ala Leu

TCG A c e CAG TGG TAC AAT GGG CGC GTT CAG CCA ACC AAT GTG TGT GTC GGG TAT C C T GTA ~ AAG A'II~ GAC ACC S e r T h r G i n T r p T y r Asn G ] y A r q V a | G i n P r o l ~ h r ASh V a l Cys V a l G I y T y r P r o V a l G I y L y s I l e Asp T h r

720

TGC cAG GGA GAC A ~ Gin

550

630

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L e u A s p I.,eu C y s

Cys

460

TGG GTG GCC GGC TGG GGA T A T ATA GAA G i G T r p V a l A l a G l y T r p G I y ql~r I l e G I u G l u

cAc ~ c

250

240

96

450

620

eta

230

270 280 290 300 310 320 330 340 --: : TTC GTC GC~ AAA AAT AAT GTG CAT GAC TGG AGA CTG GTT ~ l ~ GGA GCA AAG GAA ATT ACA TAT GGG AAC AAT AAA CCA P h e V a l G I y L y s A s h A S h V a l H i s A s p T r p A r g L e u V a l P h e GIF Ala L y s GIu I l e Thr ? y r G I y Xsn Ash L y s P r o

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ACC CCT CCC ATT TCG T G T GGG CGC T ~ Thr Pro Pro Ile Ser Cys GIy Arg Phe

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220

36

66

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360

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GTA AAA GCG CCT CTG CAA GAG AGA TAT GTG GAG AAA ATe A ~ Val Lys AIa Pro Leo Gln Glu Arg Tyr Val GIo Lys lie Ile

440

150

TAC CAC ACA TGT GGA GGC AGe TTG CTG AAT TCA CGA TGG GTG CTC ACT A~g T y r H i s T h r C y s G I y G I y S e t h e u Leu Ash Ser Arg T r p V a l L e u l~r

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260 : GCT GCT CAC TGC Ala Ala His Cys

200

6

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AGG CAA AAC CCA CAG GGT GGT GTC CGC ATC GTC GGC GGG AAG GeT GCA CAG CAT GGG GCC TGG CCC A r g G i n Asn P r o G i n G 1 y G l y V a l Arq I l e V a l G I y G I y L y s A l a A l a G i n H i s G l y A l a T r p P r o

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c ~ c cA~ A ~

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1190

1200

1210

1220

1230

1240

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9

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GCC Ala

336

366

AAC c & : eTA eAG eA~ CTC ATA GAG GTC TTG AAG GGG AAG ACC TAT TCC GAC GGA A&G AAC CAT TAT GAC &TG GAG &CC &CA GAG CTC CCA bys

Arg

Leu Gln

1250 G&A ~ Glu Leu

1360

Gln

Leu

Ile

1260 Ace Tlhr

TCG A c e Set Thr

TCC ~ A SeF *e*

Glu

Val

Leu Lys GIu

Lys Thr Tyr

Ser

Asp Gly

L y s Asn H i s

Tyr

Asp N e t G l u l~hr T h r

G l u Leu P r o

396

1270 1280 1290 1300 1310 1320 1330 1340 1350 : : : : : : : : 9 1~TGACCTGGTTCTCAACAGACCCAGTGAGCCCTTEACTCCTGAGAAAAAGGAAAGATGAAATAAAT~AATAAACATATAT~TATAGATA 402

I 370

'rACACACACACGAAAAAAA AAAAAA A

Fig. 2. The nucleotide and deduced amino acid consensus sequences of human preproacrosin. Numbering of nucleotides is given above the c D N A sequence, whereas the n u m b e r of the amino acid residues are given at the right side. The leader peptide is marked by a dotted line. The glycosylation sites are indicated by single stars. The catalytic triad of serine proteinases is formed by His-69, Asp-123 and Ser-221 (arrows). The proline-rich region extends other serine proteinases because of the presence of a C-terminal 129 amino acid domain (underlined). The stop codon is marked by a triple asterisk, and the most 3' located polyadenylation signal of A A T A A A is indicated by a solid bar

127 Homon Boor Homen Boor Homon Boor Humon Boor Humon Boer Humon Boor Humen Boor

-lOv lv lOy 20v 30v qOv HVEMLPTAILLVLAVSVVAKDNATCDGPCGLRFRONPQGGVRIVGGKAAOHGAWPWNVSLO MLPTA L V L A V S V A DNATCDGPCGLRFRO G R VGG A GAWPWNVSLO MLPTAVL-VLAVSVAARDNATCDGPCGLRFROKLESGMRVVGGMSAEPGAWPWMVSLO 50v 60v 70v 80v 90v lOOv IFTY-NSHRYHTCGGSLLNSRWVLTAAHCFVGKNNVHDWRLVFGAKEITYGNNKPVKAPLQ IF Y N RYHTCGG L L N S W V L T A A H C F K V DWRL FGA E G NKPVK PLQ [FMYHNNRRYHTCGGILLNSHWVLTAAHCFKNKKKVTDWRLIFGANEVVWGSNKPVKPPLQ 110v 120v 130v lqOv 150v 160v ERYVEKIIIHEKYNSATEGNDIALVEITPPISCGRFIGPGCLPHFKAGLPRGSQSCWVAGW ER VE I I I H E K Y S E NDIAL ]TPP CG F I G P G C L P FKAG PR 0 CMV GW ERFVEE]]]HEKYYSGLEINDIALIKITPPVPCGPF[GPGCLPOFKAGPPRAPQTCWVTGW 170v 180v 190v 200v 210v 220v GYIEEKAPRPSSILNEARVDLIDLDLCNSTOWYNGRVGPTNVCVGYPVGKIDTCGGDSGGP GY EK PR S L EARV L I D L L C N S T g N Y N G R V TNVC GYP G K I D T C G G D S G G P GYLKEKGPRTSPTLOEARVAL]DLELCNSTGMYNGRVTSTNYCAGYPRGKIDTCQGDSGGP 230v 240v 250v 260v 270v 280v LMCKDSKESAYVVVGITSWGVGCARAKRPGIYTATNPYLNWIASKIGSNALRNIQSATPPP LNC D E V V V G I T S W G V G C A R A K R P G YT T W P Y L N W I A S K I G S N A L M O TPP LMCRDRAENTFVVVGITSWGVGCARAKRPGVYTSTWPYLNWIASKIGSNALOHVOLGTPPR 290v 300v 310v 320v 330v 3qOv PTTRPPPIRPPFSHPISAHLPWYFOPPPRPLPPRPPAAOPRPPPSPPPPPPPPASPLPPPP P T PP RPP S PWYFO PP P P PRPP PP PPPPP P PPPP PSTPAPPVRPP-SVOTPVRPPWYFORPPGP--SGOPGSRPRPPAPPPAPPPPPPPPPPPPP 350v 360v 370v 380v 390v qOOv PPPPPTPSSTTKLPOGLSFAKRLOGLIEVLKGKTYS-OGKNHYDMETTELPELTSTS PPPPP K PQ L S F A K R L O g L [ E LKG S Y ETT L PPPPPPOOVSAKPPQALSFAKRLQQLIE-LKGTGNSLVERSYYETETTDLRRTARLL]

Results and discussion

Using a c D N A clone for a boar preproacrosin (Adham et al. 1989) as a probe, 8 x 105 recombinant clones for the human c D N A library were screened and three positive clones ()~HA2, )~HA6, XHA10) were obtained. A series of overlapping deletion mutants were prepared by digestion of the 1.15 kb E c o R I / E c o R I subclone with exonuclease III (Henikoff 1984). The positions of the different clones in the human preproacrosin cDNA and the sequencing strategy can be inferred from Fig. 1. The complete nucleotide sequence of 1402bp in length was constructed as a consensus sequence from 14 overlapping preproacrosin cDNAs with the help of the D N A star computer program (Fig. 2). It consists of a 20bp 5'noncoding region, which is followed by a 1263 bp open reading frame terminated by a T A G triplet and a 105 bp untranslated 3' end. The 3' untranslated region of the c D N A includes an overlapping cluster of three potential polyadenylation signals A A T A A A , the last of these being located 28 nucleotides upstream from the poly (A) tail. A comparison of the nucleotide sequence of human preproacrosin c D N A with that of the recently published boar preproacrosin c D N A (Adham et al. 1989) reveals 78% sequence identity. The human c D N A encodes a polypeptide of 421 amino acids with a calculated molecular mass of 45.9 kdalton. The respective data in the boar are 416 amino acids with a molecular mass of 45.6 kdalton. The NH2-terminus of human and boar preproacrosin is preceded by a leader sequence of 19 and 15 amino acids, respectively; this is similar to those of other transmembrane proteins (yon Heijne 1983). Thus, proacrosin is biosynthesized as a preproenzyme, preproacrosin, which is translocated through the membranes of the endoplasmic reticulum, thereby losing its leader peptide, and is then passed into the evolving acrosome of the spermatids. Immunofluorescent studies indicate that the first appearance of proacrosin is in early round spermatids during spermatogenesis (F16rke et al. 1983).

Fig. 3. Comparison of the predicted amino acid sequence of human and boar preproacrosin. Matching amino acid residues are printed between the two sequences; those that are negatively related are shown as a blank. Gaps indicated as dashes are introduced to maximize homology of pairing amino acids

The amino acid sequences of human and boar preproacrosin deduced from the respective c D N A inserts are compared in Fig. 3 and are 70% homologous over the entire sequence. The homology increases to 81% in the leader peptide. There is one domain that exhibits great differences in the amino acid sequence between human and boar preproacrosin; it has not previously been found in any other serine proteinase. This C-terminal domain contains 127 amino acids in the boar and 129 amino acids in the human, and is extremely rich in proline (42 in the boar, 45 in the human). The homology in this domain between human and boar is 56.1%. It has been suggested that this proline-rich domain is processed during the autodigestion of ct-acrosin to [3-acrosin (Zelezna and Cechova 1982), which is the enzymatically active and more stable acrosin form. However, there is an even more attractive hypothesis with respect to the function of the proline-rich domain. If acrosin is not only involved in sperm penetration through the zona pellucida (McRorie and Williams 1974), but also in the recognition and binding of the sperm to the zona pellucida (Jones et al. 1988; TBpfer-Petersen and Henschen 1988), then the acrosin molecule must be species specific. Because of the extensive differences in the amino acid sequences of the prolinerich domain between human and boar preproacrosin, we assume that this domain reflects the species-specificity of acrosin. The three active site residues essential for the proteolytic activity of serine proteinases, namely histidine, aspartic acid and serine, are found in human and boar preproacrosin in conserved positions (human: His 69, Asp 123, Ser 221; Boar: His 70, Asp 124, Ser 222). Furthermore, the possible-N-linked glycosylation attachment sites (human: Asn 3, Asn 191; boar: Asn 3, Asn 192) and the positions of the 12 cysteines responsible for crosslinking in the acrosin molecule are positionally conserved in human and boar preproacrosin. As compared with other serine proteinases where the positions of the disulfide bonds are known (Young et al. 1978), 8 cysteine residues in human preproacrosin can be predicted to crosslink the heavy chain (Cys 54-70; Cys 158-227; Cys 190-206; Cys 217-247).

128 Acknowledgements. We gratefully acknowledge the technical assis-

tance of Ina Ebrecht, Ute Schrader, Christiane Schmidt and Jutta Sist. This work was supported by the Deutsche Forschungsgemeinschaft (En 84/18-3).

References

Fig.4. Northern blot analysis. Total RNA (20 gg) from testes of 11 mammalian species was electrophoresed, blotted onto nitrocellulose filters and hybridized with the nick-translated human preproacrosin cDNA probe. The hybridizing band is identical in all species and was estimated to be approximately 1.6 kb by comparison with RNA standards (Bethesda Research Laboratories); lane 1 human, lane 2 Rhesus monkey, lane 3 bull, lane 4 boar, lane 5 sheep, lane 6 goat, lane 7 Guinea pig, lane 8 rabbit, lane 9 Russian hamster, lane 10 mouse, lane 11 rat

The other two disulfide bonds that join the light and the heavy chains are b e t w e e n cysteines 10 and 6 of the light chain and cysteines 135 and 143 of the heavy chain. There is wide variation in the reported molecular mass of human proacrosin as d e t e r m i n e d by S D S - P A G E ; it ranges between 75 kdalton and 47 kdalton (Mack and Zaneveld 1986). H o w e v e r , the molecular mass of the proacrosin polypeptide, as determined from the amino acid sequence deduced from the c D N A sequence, can be calculated to be 43.9 kdalton. A similar difference was found for boar proacrosin (Polakoski and Parrish 1977; A d h a m et al. 1989); this could be caused, in part, by the glycosylation of proacrosin, which contains two potential glycosylation sites. Glycosylation seems to be a general attribute of m a m m a l i a n proacrosin and acrosin. Rabbit proacrosin (Mukerji and Meizel 1979) and the acrosin of boar (Fock-Nt~zel et al. 1984) and goat (Hardy et al. 1989) have been found to be glycosylated, e.g., 8% carbohydrate by weight in boar acrosin. The transcripts of preproacrosin of the human and of 10 other mammalian species are identical in length, around 1.6kb, as shown in Fig. 4. If one excludes the leader sequence, which should be a general c o m p o n e n t of the mammalian preproacrosin and is absent in the mature proacrosin polypeptide, the molecular mass of the proacrosin polypeptide of all mammals studied can be determined to be around 44 kdalton. In contrast, considerable variation in the molecular mass of mature proacrosin purified from testes and spermatozoa of different m a m m a l s has been observed. It ranges from 75 to 47 kdalton in the h u m a n (Mack and Z a n e v e l d 1986), 68 kdalton in the rabbit (Mukerji and Meizel 1979), 55-53 kdalton in the boar (Polakoski and Parrish 1977), 48 kdalton in the bull (Elce and M c I n t y r e 1983), and 43-62 kdalton in guinea pig (Hardy et al. 1987). E v e n if one considers the glycosylation of the proacrosin polypeptide in the different species, the differences between the molecular weights mainly determined by SDSP A G E and those calculated from Northern-blot analysis remain inexplicable. The discrepancy could be a result of the anomalous electrophoretic behavior of the glycosylated proacrosin or of inadequate attention being paid to the oxidation state of the acrosin samples and/or standards.

Adham IM, Klemm U, Maier W-M, Hoyer-Fender S, Tsaousidou S, Engel W (1989) Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis. Eur J Biochem 182 : 563-568 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 Elce JS, McIntyre EJ (1983) Acrosin: immunochemical demonstration of multiple forms generated from bovine and human proacrosin. Can J Biochem Cell Biol 61 : 989-995 F16rke S, Phi-van L, M~ller-Esterl W, Scheuber HP, Engel W (1983) Acrosin in the spermiohistogenesis of mammals. Differentiation 24 : 250-256 Fock-Nfizel R, Lottspeich F, Henschen A, Mtiller-Esterl W (1984) Boar acrosin is a two-chain molecule. Isolation and primary structure of the light chain; homology with the pro-part of other serine proteinases. Eur J Biochem 141:441-446 Hardy DM, Wild GC, Tung KS (1987) Purification and initial characterization of proacrosins from guinea pig testes and epididymal spermatozoa. Biol Reprod 37 : 189-199 Hardy DM, Shoots AFM, Hedrick JL (1989) Caprine acrosin. Purification, characterization and proteolysis of the porcine zona pep lucida. Biochem J 257 : 447-453 Heijne G von (1983) Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133 : 17-21 Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359 Jones R, Brown CR, Lancaster RT (1988) Carbohydrate-binding properties of boar sperm proacrosin and assessment of its role in sperm-egg recognition and adhesion during fertilization. Development 102 : 781-792 Mack SR, Zaneveld LJD (1986) Comparative activation studies with extracted and purified human proacrosin. Comp Biochem Physiol [B] 83 : 537-543 Maxam AM, Gilbert W (1980) Sequencing end labeled DNA with base specific chemical cleavages. Methods Enzymol 65 : 499-560 McRorie RA, Williams WL (1974) Biochemistry of mammalian fertilization. Annu Rev Biochem 43 : 777-803 Mtiller-Estelr W, Fritz H, Fock-Niizel R, Lottspeich F, Henschen A (1984) Structure, function and biosynthesis of the sperm proteinase acrosin. In: Voelter W, Bayer E, Ovchinnikov YA, Wt~nsch E (eds) Chemistry of peptides and proteins, de Gruyter, Berlin New York, pp 377-386 Mukerji SK, Meizel S (1979) Rabbit testis proacrosin. Purification, molecular weight estimation, amino acid and carbohydrate composition of the molecule. J Biol Chem 254:11721-11728 Polakoski KL, Parrish RF (1977) Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. J Biol Chem 252 : 1888-1894 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 T6pfer-Petersen E, Henschen A (1988) Zona pellucida-binding and fucose-binding of boar sperm acrosin is not correlated with proteolytic activity. Biol Chem Hoppe-Seyler 369 : 69-76 Young CL, Barker WC, Tomaselli CM, Dayhoff MO (1978) Serine proteases. In: Dayhoff MO (ed) Atlas of protein sequences and structure, vol 5. NBR Foundation, Washington, DC, pp 73-93 Zelezna B, Cechova D (1982) Boar acrosin. Isolation of two active forms from boar ejaculated sperm. Hoppe-Seyler's Z Physiol Chem 363 : 757-766

Received June 1, 1989 / Revised July 24, 1989