Amidolytic activity of prostatic acid phosphatase on humansemenogelins and semenogelin-derived synthetic substrates MicheÁle Brillard-Bourdet1, Sophie ReÂhault1, Luiz Juliano2, MicheÁle
Trang 1Amidolytic activity of prostatic acid phosphatase on human
semenogelins and semenogelin-derived synthetic substrates
MicheÁle Brillard-Bourdet1, Sophie ReÂhault1, Luiz Juliano2, MicheÁle Ferrer1, Thierry Moreau1
and Francis Gauthier1
1 Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University FrancËois Rabelais, Faculty of Medicine, Tours, France; 2 Departamento de BiofõÂsica, Escola Paulista de Medicina, Universidade Federal de SaÄo Paulo, SaÄo Paulo, Brazil
In addition to kallikrein hK3, a serine protease generally
reported as PSA (prostate-speci®c antigen), at least two
other enzymes in human seminal plasma also cleave
syn-thetic peptidyl substrates derived from the sequence of
human semenogelins We have identi®ed one of these as
prostatic acid phosphatase (PAP), a major component of
prostatic ¯uid whose physiological function is unclear The
other is a high Mrbasic protein present at low concentrations
in seminal plasma and that remains to be characterized PAP
was puri®ed to homogeneity from freshly ejaculated seminal
plasma Its N-terminal sequence and its phosphatase
prop-erties (hydrolysis of para-nitrophenylphosphate at low pH)
were determined, and its inhibition by sodium ¯uoride
measured Both puri®ed and commercial PAP also had
amidolytic activity on peptide substrates derived from the
semenogelin sequence at neutral and slightly basic pH The
kcat/Km values were in the 102)103 M)1ás)1 range using
¯uorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identi®ed
ex vivo PAP cleavage sites diered from those of hK3 and were mainly at P1 Gln residues or between residues bearing hydroxyl groups PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors Only 3±4 dichloroisocoumarin and benzamidine inhibited puri®ed PAP Puri®ed human seme-nogelin was cleaved by puri®ed and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identi®ed ex vivo by analysis of seminal plasma
Keywords: amidolytic activity; ¯uorogenic substrates; human kallikrein; phosphatase; semenogelins
The semenogelins I and II are secreted by the seminal
vesicles into human semen where they immediately form a
coagulum that entraps spermatozoa upon ejaculation This
coagulum dissolves within a few minutes after ejaculation as
a result of proteolysis involving kallikrein hK3
(prostate-speci®c antigen, PSA) [1,2] This appears to be the main
physiological function of this protease, although it may act
on several other biological substrates, such as insulin-like
growth factor binding proteins [3±5] and parathyroid
hormone-related protein [6] Ex vivo analysis by
two-dimensional electrophoresis of fresh ejaculates identi®ed
preferential cleavage sites in semenogelins at Tyr136, Tyr292
and Gln266 of semenogelin I (S ReÂhault & M
Brillard-Bourdet,
3 unpublished data) All three cleavage sites have
been identi®ed by in vitro studies [7], the third one only very
recently [8] We prepared peptidyl ¯uorogenic substrates of
hK3 based on these sequences and on others containing
previously identi®ed cleavage sites, in an attempt to obtain
more sensitive substrates of hK3 than those that are available commercially Some of them were cleaved by at least two other proteases obtained by ion exchange chro-matography from crude seminal plasma This study shows that one of these enzymes is prostatic acid phosphatase (PAP), although this enzyme has never been attributed any proteolytic or amidolytic activity to date Large amounts of PAP (1 mgámL)1) are present in seminal ¯uid [9], but its physiological function remains unclear even its cellular form has neutral protein-tyrosine kinase phosphatase activity [10]
We have puri®ed PAP to homogeneity and compared its amidolytic properties to those of commercial source using semenogelin-derived ¯uorogenic and puri®ed semenogelins
M A T E R I A L S A N D M E T H O D S
Materials The following materials were obtained from commercial sources: DEAE±Sephadex A50, Sephacryl S300, Phenyl-Sepharose (Pharmacia), NaF, (Octylphenoxy) poly-ethoxyethanol (IGEPAL
dimethyl-formamide, acetonitrile (Merck), CF3COOH
Elmer) All other reagents were of analytical grade Com-mercial PAP puri®ed from human semen was from Sigma Puri®cation and characterization of PAP
Approximately 50 mL of seminal plasma was obtained from healthy donors, dialysed against 3 ´ 2 L 25 mMTris/HCl
Correspondence to F Gauthier, Laboratory of Enzymology and
Protein Chemistry, INSERM EMI-U 00-10, University FrancËois
Rabelais, Faculty of Medicine, 2 bi, Boulevard TonnelleÂ,
37032 Tours cedex France Fax: + 33 2 47 36 60 46,
E-mail: gauthier@univ-tours.fr
Abbreviations: PSA, prostate-speci®c antigen; PAP, prostatic acid
phosphatase; DCI, 3-4 dichloroisocoumarin
(octylphenoxy) polyethoxyethanol; Abz, O-aminobenzoyl;
EDDnp, ethylenediamine 2,4-dinitrophenyl.
(Received 23 July 2001, revised 31 October 2001,
accepted 9 November 2001)
Trang 2pH 7.8, 0.15MNaCl, and fractionated on DEAE A50 The
column (2.6 ´ 40 cm) was washed with 200 mL equilibrium
buffer and proteins were eluted at 28 mLáh)1 with a
discontinuous gradient of NaCl (25 mM, 80 mM, and
200 mM) Fractions were assayed for their enzymatic
activity towards Abz-ISYQSSSTEEQ-EDDnp as reported
under Kinetic measurements Fractions that were eluted
from DEAE A50 with 200 mM NaCl were fractionated
further on Sephacryl S300 The column (2.6 ´ 100 cm) was
equilibrated in the same buffer as before and eluted at
60 mLáh)1 Fractions of the second eluted peak were
pooled, checked for activity towards the same substrate as
before, and run onto a phenyl-Sepharose column (1 ´ 7 cm)
equilibrated with 25 mM Tris/HCl pH 7.8, 1.5M (NH4)2
SO4 The column was eluted with a decreasing
discontin-uous gradient of ammonium sulfate Enzymatically active
fractions were concentrated and checked for purity by four
methods First, HPLC on C4 (C4 uptisphere column, 5 lm,
30 mm ´ 2.1 mm) eluted with a linear gradient of
aceto-nitrile (0±60%, v/v) in 0.075% CF3COOH for 40 min at a
¯ow rate of 0.2 mLámin)1; second MALDI-TOF MS using
a BruÈcker Re¯ex mass spectrometer; third SDS/PAGE and
fourth N-terminal sequencing The protein concentration
was calculated from a BCA assay and the activity is
expressed with reference to the phosphatase activity of
commercial PAP
Puri®cation of human semenogelins
Semenogelins were puri®ed essentially as described by
Malm et al with some modi®cations [11] Ejaculates from
healthy volunteers were collected on DEAE cellulose A50
equilibrated in 40 mMTris/HCl pH 9.7 in the presence of
4M urea and a cocktail of inhibitors There was no
signi®cant degradation of semenogelins under these
condi-tions, which allow the rapid separation by centrifugation of
cellular components and gel-bound proteases The ®nal step
of puri®cation was HPLC fractionation on a C4 column
using a 0±60% (v/v) linear gardient of acetonitrile in 0.075%
CF3COOH for 40 min at a ¯ow rate of 0.2 mLámin)1
Synthesis of ¯uorogenic substrates
The intramolecularly quenched ¯uorogenic peptides were
synthesized by solid-phase method; glutamine was the
C-terminal residue of all peptides due to a requirement of
the synthesis strategy [12] All syntheses were performed
with Fmoc methodology using a multiple automated
peptide synthesizer (PSSM-8, Shimadzu Co.)
Intramolec-ularly quenched ¯uorogenic substrates were prepared as
2 mM stock solutions in dimethylformamide and diluted
with activation buffer Substrate purity was checked by
MALDI-TOF MS (TofSpec-E, Micromass) and by
re-versed-phase chromatography on a C18 column eluted with
a 10-min linear gradient of 0±60% acetonitrile in 0.075%
CF3COOH at 0.5 mLámin)1
Kinetic measurements
All amidolytic assays were carried out at 37 °C in 20 mM
Tris/HCl pH 9.0 Speci®city constants (kcat/Km) were
determined for Abz-peptidyl-EDDnp substrates under
pseudo-®rst order conditions, using a substrate
concentra-tion far below the Km, as reported elsewhere [13] Calcula-tions were done using ENZFITTER software (Biosoft) Excitation and emission wavelengths were 320 nm and
420 nm for experiments with intramolecularly quenched
¯uorogenic substrates The system was standardized using FR-OH prepared by total tryptic hydrolysis of Abz-FR-pNA, and its concentration was determined from the absorbance at 410 nm, assuming e410nm 8800M)1ácm)1 for p-nitroanilide Substrate concentrations of Abz-pept-idyl-EDDnp were determined by measuring the absor-bance at 365 nm, assuming e365nm 17 300M)1ácm)1for EDDnp Kms were measured using six substrate concentra-tions (0.2±12 lM), the ®nal concentration of PAP was in the 0.1 lMrange The phosphatase activity of the puri®ed PAP was measured using p-nitrophenylphosphate as substrate and the method recommended by the manufacturer (Sigma) This activity was compared to that of commercial PAP and the concentration of active enzyme calculated on this basis
The pH activity pro®les were determined by measuring the phosphatase and the peptidase activities of puri®ed PAP over the pH range 3±10 at 0.5 unit intervals at 37 °C 20 mM citrate/phosphate buffers were used over the pH range 3±7,
20 mMphosphate buffer for pH 7±8, 20 mMTris/HCl for
pH 8±9 and 20 mMcarbonate/bicarbonate buffer for pH 9±
10 All other experimental conditions were as reported above
Inhibition of puri®ed PAP The amidolytic activity of puri®ed PAP (60 nM®nal) was measured in the presence of a molar excess of inhibitors from all four classes of proteases ) aprotinin, benzamidine, leupeptin, Soya bean trypsin inhibitor (SBTI)
(1-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E64
o-phenanthroline, and NaF, which inhibits phosphatase activity [14] Inhibitors were mixed with the enzyme for
15 min in 20 mM Tris/HCl pH 9.0, 0.05% IGEPAL at
37 °C, before adding the ¯uorogenic substrate (2 lM) Inhibition is expressed with reference to an inhibitor-free control incubated under the same experimental conditions Conversely, acid phosphatase activity was measured in the presence of NaF and of those of inhibitors that alter amidolytic activity Incubation with inhibitors was per-fromed at pH 9 and at pH 4.8, before the PAP activity was measured at pH 4.8 The in¯uence of ¯uorogenic substrate binding on phosphatase activity was also assayed by incubating PAP with Abz-SSIYSQTEEQ-EDDnp (2 lM and 20 lM ®nal) under the same experimental conditions and then measuring phosphatase activity
Identi®cation of cleavage sites in peptidyl substrates Fluorogenic peptides (15±30 lM®nal) were incubated with puri®ed PAP in the 0.1 lM range at 37 °C in 20 mM Tris/HCl pH 9.0 containing 0.02% IGEPAL Aliquots (200 lL) were removed at intervals up to 60 min and the reaction was blocked by adding 4 lL CF3COOH The samples were fractionated on a C18 cartridge column (5 lm, 30 mm ´ 2.1 mm) using a linear gradient of acetonitrile (0±60%, v/v) in 0.075% CF3COOH for
10 min at a ¯ow rate of 0.5 mLámin)1, with simultaneous recordings at three wavelengths (220, 320, and 360 nm)
Trang 3This allowed direct identi®cation of EDDnp-containing
peptides prior to N-terminal sequencing of all signi®cant
peaks
Amino acid sequence analysis of peptide products
The amino-acid sequences were determined using an
Applied Biosystems 477A pulsed liquid sequencer with the
chemicals and program recommended by the manufacturer
Phenylthiohydantoin derivatives were identi®ed using an on
line model 120A PTH analyser
R E S U L T S A N D D I S C U S S I O N
Sperm lique®es very rapidly after ejaculation due to the
proteolytic cleavage of the coagulum-forming proteins
semenogelins I and II This results in the progressive release
of motile spermatozoa and also in the generation of
biologically active semenogelin fragments, the function of
which is not yet fully understood [7,15] Kallikrein hK3 has
long been described as the protease responsible for this
proteolysis, based on its high concentration in seminal
plasma and on its chymotrypsin-like speci®city It thus
cleaves puri®ed semenogelins mainly at tyrosine residues,
although cleavage may also occur at histidine and glutamine
residues after prolonged incubation of puri®ed semenogelins
with hK3 [7,8] However, total ex vivo hydrolysis of
semenogelins is completed within minutes following
ejacu-lation, suggesting that some other proteases participate to
this process We have prepared an intramolecularly
quenched ¯uorescent substrate (Abz-ISYQSSSTEEQ
EDDnp) that encompasses a sequence containing a major
ex vivo cleavage site, the Gln266±Ser267 bond of
semenog-elin I (S ReÂhault & M Brillard-Bourdet, unpublished
data)
8 This cleavage site has been also recently identi®ed in
puri®ed semenogelins after extensive cleavage by hK3 [8],
although it was initially reported to be one residue upstream
in this sequence, at the Tyr265±Gln266 bond [7] Hydrolysis
of this substrate by samples from the fractionation of
seminal plasma on an anion exchanger revealed two
fractions that cleaved this peptide, one eluting with the
ionic strength used for PSA/hK3, and the other, with a
much lower pHi, with the buffer containing 200 mMNaCl
(Fig 1A) This second peak was fractionated further by gel
chromatography (Fig 1B), to give two peaks with
amido-lytic activity, one was eluted in the void volume and the
other in the 50±100 kDa fractions Semen thus contains at
least three proteases that have amidolytic activity on this
semenogelin-derived substrate They had different substrate
speci®cities; hK3 cleaved at the tyrosine±glutamine bond,
whereas the Sephacryl S300-excluded fraction cleaved at the
glutamine±serine bond and the ®ltered fraction at the
serine±tyrosine bond, as shown by HPLC fractionation and
N-terminal sequencing Part of the last ®ltered fraction was
puri®ed further by hydrophobic chromatography on
phe-nyl-Sepharose Fractions with enzymatic activity were
analysed by MALDI TOF MS and reversed-phase HPLC
on a C4 cartridge A molecular mass of 47 798 Da was
determined by MS and a single, symetrical peak was eluted
from the C4 column (Fig 1C) The collected fraction was
immediately equilibrated in neutral buffer to determine
residual peptidase activity Its N-terminal sequence (20
residues) was 100% identical to PAP and showed no trace
of contamination This fraction still had enzymatic activity
on Abz-ISYQSSSTEEQ-EDDnp when buffered at neutral
pH immediately after elution from the C4 cartridge, demonstrating that the sequenced product and that with peptidase activity were the same The puri®ed enzyme was checked for its phosphatase activity on p-nitrophenyl phosphate and this activity was compared to that of commercial PAP Based on a molar concentration of about
9 lM estimated from a BCA protein assay for the PAP monomer, a value of 1.67 phosphatase unitsánmol)1was calculated for puri®ed PAP, which is in the same range as that reported for commercial PAP Con®rmation that PAP had amidolytic activity was obtained by showing that commercial PAP hydrolysed the Abz-ISYQSSSTEEQ-EDDnp substrate at neutral pH, as does the puri®ed enzyme The ratio of the amidolytic activity of puri®ed to that of commercial PAP (2.5) was also in agreement with that obtained by measuring phosphatase activity (1.75) PH-dependence of PAP amidolytic activity
The pH-dependence of PAP amidolytic activity was studied over the pH range 3±9 The Abz-ISYQSSSTEEQ-EDDnp substrate was not hydrolysed at acidic pH, at which phosphatase activity is greatest (Fig 2) There was amido-lytic activity at neutral pH, with a maximum at about
pH 9.0
Inhibition of PAP amidolytic activity
A series of inhibitors of all classes of enzymes were tested for their ability to inhibit PAP Only 3±4 dichloroisocoumarin (DCI;
9 200 lM ®nal) and benzamidine (0.5 mM ®nal) partially inhibited PAP amidolytic activity under the experimental conditions used Pepstatin, leupeptin, PhCH2SO2F, SBTI, E64 and EDTA did not signi®cantly inhibit PAP amidolytic activity However, NaF (10 mM), which strongly inhibits phosphatase activity at acidic pH, inhibited PAP amidolytic activity at neutral pH
Converse-ly, DCI and benzamidine partially inhibited phosphatase activity (data not shown)
Incubating the substrate Abz-SSIYSQTEEQ-EDDnp with puri®ed or commercial PAP at basic or acidic pH also resulted in the inhibition of phosphatase activity, demon-strating that the ¯uorogenic substrate, although not cleaved
at pH 4.6, binds to the enzyme active site under these conditions
PAP amidolytic activity on semenogelin-derived substrates
The amidolytic activity of PAP was assayed using four
¯uorogenic substrates based on the ex vivo cleavage sites of semenogelin I (S ReÂhault & M Brillard-Bourdet, unpub-lished data)
10 Those sites are at Tyr136, Tyr292 and Gln326 and all three have been identi®ed as putative hK3/PSA cleavage sites [8] Two of these substrates included the Gln326±Ser327 site, but within two different peptides corresponding to residues 321±329 (substrate 3) or 323±
332 (substrate 4) of the semenogelin I sequence The peptide containing Tyr136 (peptide 1) was cleaved at the glutamine± tyrosine bond by puri®ed PAP and the peptide containing Tyr292 (peptide 2) was cleaved at the glutamine±threonine
Trang 4bond, with a second cleavage after tyrosine PAP also preferentially cleaved after a glutamine residue, at the glutamine±serine bond in Abz-NKISYQSSSQ-EDDnp, and secondarily at tyrosine±glutamine (Table 1) Speci®city constants (kcat/Km) were measured under ®rst-order condi-tions (Table 1) They indicated a rather low cleaving ef®ciency, even though there was a tight interaction between PAP and Abz-peptidyl-EDDnp substrates, as deduced from the low values of Kmin the micromolar range for substrate 1 (0.23 lM) and substrate 4 (4 lM) which are the only two having a single cleavage site
A shift in PAP speci®city was observed depending on the length and/or the nature of the peptidyl moiety in the substrate This occurred with substrate 4, whose cleavage site is shifted towards the serine±tyrosine bond when the N-terminal asparagine±lysine dipeptide was removed from substrate 3 This phenomenon, which demonstrates the in¯uence of surrounding subsites on P1 occupancy, was also reported by Coombs et al [16] for hK3/PSA hydrolysing
Fig 1 Puri®cation of prostatic acid phosphatase from human seminal plasma (A) SDS/PAGE of the 200 m M NaCl elution from the DEAE A50 column equilibrated in 25 m M Tris/HCl pH 7.8 Lane 2 shows a sample from the 80 m M NaCl elution containing hK3/PSA (M r(app) 33 kDa) (B) The 200 m M NaCl-eluted fractions were pooled, fractionated on Sephacryl S300, checked for their amidolytic activity (dashed line) and those corresponding to the shaded area (lanes 1±3 on SDS/PAGE) were puri®ed further by hydrophobic chromatography on phenyl-Sepharose (C) Phenyl-Sepharose fractions were pooled and checked for purity by SDS/PAGE and C4 reversed-phase chromatography using a gradient of 0±60% acetonitrile in 0.075% CF 3 COOH.
Fig 2 pH activity pro®le of PAP activity (full line) and PAP amidolytic
activity (dashed line) Results are normalized rates and are the means of
two experiments at each pH.
Trang 5peptide substrates Nevertheless the preferential cleavage
sites for PAP in the peptides used here were with glutamine
at P1 and hydroxyl-bearing (Ser, Thr, Tyr) residues at P1¢
We do not know why PAP has amidolytic activity at neutral
pH But its speci®city differs from that of hK3/PSA, which
also cleaves these substrates, but at different sites (S ReÂhault
& M Brillard-Bourdet, unpublished data)
con®rmed that enzymatic activity of the PAP preparation is
not due to the presence of trace amounts of hK3 by showing
that: (a) the hK3 substrate Suc-AAPF-pNa is not cleaved
by PAP at a 1 lM ®nal concentration; (b) a polyclonal
antibody against PSA does not reveal any band after
immunoblotting; (c) the amidolytic activity is strictly
associated with the peak of PAP after reversed-phase
HPLC, and no trace of contaminant at the elution time of
PSA was observed; (d) commercial PAP has amidolytic
activity similar to that of puri®ed PAP
The fact that there was a shift in the cleavage of synthetic
peptides by PAP raised the question of whether PAP cleaves
a protein substrate such as semenogelin, which also
questions the biological relevance of PAP amidolytic
activity We therefore measured the ability of puri®ed and
of commercial PAP to cleave freshly prepared
semeno-gelin I, and we identi®ed the main cleavage sites in the
protein
PAP proteolytic activity on puri®ed semenogelins
Semenogelins were puri®ed from seminal plasma in such a
way that no proteolysis could occur upon sample collection
Puri®ed semenogelin I was checked for purity by
reversed-phase HPLC, and used as a substrate for puri®ed and for
commercial PAP PAP (1 lM ®nal) was incubated with
puri®ed semenogelin 1 (16 lM ®nal) for 18 h at 37 °C in
20 mMTris/HCl pH 9.0, 0.05% IGEPAL The mixture was
then fractionated by HPLC on a C4 column, and the
cleavage sites identi®ed by N-terminal sequencing (Fig 3)
Two cleavage sites were identi®ed, one at Ser170 and the
other at Tyr292 The former has not been described before,
whereas the latter is a major cleavage site for hK3 [8]
Tyr292 is also present in substrate 2 used in this study
(Table 1) It is cleaved by PAP at the Tyr292±Ser bond and
at the Gln294±Thr bond, suggesting that accessibility to the
sensitive bond is different in the peptide substrate and in the
protein
Semenogelin was hydrolysed by a PAP concentration
lower than that in the 10)5 M range found in semen [9]
Only a few sites were identi®ed however, and the extent of proteolysis was lower than that obtained with hK3 under similar conditions (data not shown) Nevertheless, pros-tatic PAP could be involved in semenogelin processing and degradation because of its high concentration in seminal plasma, its rather wide substrate speci®city, with cleavage after glutamine or hydroxylated residues, and its relatively slow inactivation in semen The rapid hydrolysis
of semenogelin by hK3/PSA gives large fragments that could be further processed by PAP, which is stable as a protease at neutral pH, and whose phosphatase activity requires an acid environment This condition occurs physiologically only when the enzyme reaches the female genital tract [17]
A C K N O W L E D G E M E N T S
The authors are indebted to C BartheÂleÂmy and M H Saussereau (Department of Human Reproduction CHRU Bretonneau, Tours) for their collaboration and helpful advice, and in Brazil to the FundacËaÄo de Amparo a Pesquisa do Estado de SaÄo Paulo (FAPESP) The English text was checked by Amanuensis Scienti®ca.
R E F E R E N C E S
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3 Rehault, S., Monget, P., Mazerbourg, S., Tremblay, R., Gutman, N., Gauthier, F & Moreau, T (2001) Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3 Eur J Biochem 268, 2960± 2968.
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Fig 3 Hydrolysis of puri®ed semenogelin I by PAP HPLC fraction-ation on C4 of semenogelin incubates with (solid line) or without (dashed line) puri®ed PAP All major fractions were sequenced N-terminally to identify two cleavage sites, one at Ser170 (peak 1) and one at Tyr292 (peak 2) The main peak on each chromatogram was the IGEPAL and the last peak eluted was PAP.
Table 1 Second order rate constants (k cat /K m ) for the hydrolysis of
semenogelin-derived synthetic peptidyl substrates by puri®ed prostatic
acid phosphatase k cat /K m values were determined under pseudo-®rst
order conditions The results are the mean of two recordings for each
substrate Cleavage sites are identi®ed after numbered residues in
peptide sequences Numbers refer to the positioning of the residue in
the human smenogelin I sequence.
Substrate k cat /K m ( M)1ás )1 )
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