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Journal ArticleDOI

A Refined Kinetic Analysis of Plasminogen Activation by Recombinant Bovine Tissue-Type Plasminogen Activator Indicates Two Interconvertible Activator Forms†

19 Aug 1998-Biochemistry (American Chemical Society)-Vol. 37, Iss: 36, pp 12631-12639

TL;DR: A refined new method of kinetic analysis is proposed which allows examination of both stationary and prestationary phases of this process and revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio.

AbstractBovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

Summary (2 min read)

Introduction

  • The activation of plasminogen is an important process associated with degradation of an extracellular matrix such as dissolving of blood clots, tissue remodeling, and invasive growth of cancer cells.
  • Tissue-type plasminogen activator (tPA)1 and urokinase-type plasminogen activator (uPA) are the two major proteins responsible for conversion of plasminogen to plasmin.
  • The tPA-induced process is stimulated significantly on the surface of fibrin, and tPA is regarded as the fibrinolytic activator.
  • Kinetic analysis of the human tPA catalysis was rendered in a number of papers (7-9) and resulted in several conclusions concerning its mechanism.
  • This work is part of the FØTEK program supported by the Danish Dairy Research Foundation (Danish Dairy Board) and the Danish Government.

MATERIALS AND METHODS

  • P. pastorisGS115 (his4) (10), the protease deficient strain SMD1168 (his4, pep4), and the expression vectors pPIC9K and pHIL-D2 were purchased from Invitrogen Corp.
  • Sequencing, ligation, transformation ofEscherichia coli, DNA preparation, PCR, and other DNA modifying processes were performed according to the manufacturers’ recommendations or standard laboratory procedures.
  • To generate the native N-terminus of tPA after cleavage by the signal peptidases , site specific mutagenesis was performed on pPIC9K/tPA, using the primer 5′-CTCGAGAAAAGAGAGGCTGAAGCTTCGTACAAAGTGACCTGCAGAGAT-3′.
  • The plasminogen activation potential of tPA was evaluated in a coupled peptidyl anilide assay, where the formation of plasmin was measured by its hydrolysis of the chromogenic substrate S-2251.
  • A more complex scheme, described in model 2, considers the existence of a prestationary step before the above reactions which distorts linearity of the plot in its initial part.

RESULTS

  • The expression level of bovine tPA was optimized by a 3-fold strategy: (1) improving the secretion, (2) increasing the transcription of the tPA gene, and (3) minimizing the proteolytic breakdown of tPA in the expression media.
  • Second,∼10 000 colonies were screened on YPD plates containing different concentrations of G418 for multicopy colony selection.
  • The band at approximately 50 kDa turned out to be the proteinase domain, even though it was 10-20 kDa higher than the calculated mass (29 kDa).
  • (2) The reaction was performed with a decreased concentration of tPA (1 × 10-3 µM) in the presence of fibrinogen fragments.
  • At the same time, analysis of the prestationary kinetics from the same experiment revealed the presence of a tPA form with high affinity to Pg being in equilibrium with a low affinity form, see model 2.

DISCUSSION

  • The authors have produced recombinant bovine tissue-type plasminogen activator in the yeast,P. pastoris.
  • The equilibrium between these forms in the preparation was shifted in favor of tPA* (g50:1).
  • At the same time, it showed a≈25-fold increase ofkcat, when compared to the Fb-free tPA.
  • The mechanism of activation of human tPA by fibrinogen fragments is presumably the same as for bovine tPA according to the values of calculated rate constants.
  • To characterize other properties of the produced tPA, inhibition of bovine single-chain tPA by PAI-1 and PAI-2 was investigated.

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A Refined Kinetic Analysis of Plasminogen Activation by Recombinant Bovine
Tissue-Type Plasminogen Activator Indicates Two Interconvertible Activator
Forms
Laust B. Johnsen,
§
Peter Ravn,
§,
Lars Berglund,
§
Torben E. Petersen,*
Lone K. Rasmussen,
§
Christian W. Heegaard,
§
Jan T. Rasmussen,
§
Connie Benfeldt,
|
and Sergey N. Fedosov
§
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, UniVersity of Aarhus, Science Park,
GustaV Wieds Vej 10 C, DK-8000 Aarhus C, Denmark, and MD Foods Research and DeVelopment Centre, RørdrumVej 2,
DK-8220 Brabrand, Denmark
ReceiVed March 23, 1998; ReVised Manuscript ReceiVed June 18, 1998
ABSTRACT: Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylo-
trophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-
mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments.
We have proposed a refined new method of kinetic analysis which allows examination of both stationary
and prestationary phases of this process. The investigation revealed the presence of two interconvertible
forms of the recombinant bovine tPA being in equilibrium ata1to50ratio. Only the minor form was
able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen
concentration (K
m
g 5µM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments
activated the single-chain tPA due to preferential binding and stabilization of the minor “active” form of
the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to
human tPA as well. The K
m
values, obtained for recombinant bovine and human tPA in the presence of
fibrinogen fragments, were found to be similar (K
m
) 0.1 µM) while k
cat
of human tPA was 5-10 times
higher.
The activation of plasminogen is an important process
associated with degradation of an extracellular matrix such
as dissolving of blood clots, tissue remodeling, and invasive
growth of cancer cells. Tissue-type plasminogen activator
(tPA)
1
and urokinase-type plasminogen activator (uPA) are
the two major proteins responsible for conversion of plas-
minogen to plasmin. Although both tPA and uPA are related
enzymes and activate plasminogen by cleavage of the same
peptide bond, they have their own physiological features.
The tPA-induced process is stimulated significantly on the
surface of fibrin, and tPA is regarded as the fibrinolytic
activator. On the other hand, the pericellular uPA-mediated
activation of plasminogen is supposed to be engaged in tissue
remodeling and cancer metastasis development.
The activation of plasminogen has been studied in detail
in the human system from where the involved protein
components have been identified and characterized. In
contrast much less is known about plasminogen activation
in other species. Bovine mastitis is an inflammatory disease
of the mammary gland induced by various microorganisms,
and a 20-fold increase in tPA activity has been reported in
the milk of cows infected with Staphylococcus aureus (1).
The activation of plasminogen by tPA is greatly increased
by the R
s2
-casein dimer (2), and in order to study this system
in more detail it is necessary to obtain bovine tPA. As this
protein is only present in very small amounts in natural
tissues and fluids and to our knowledge never has been
purified, we have made recombinant expression of the
protein.
tPA is a mosaic protein with five domains consisting of a
finger domain, an epidermal growth factor domain, two
kringle domains, and a serine proteinase domain. The finger
and the second kringle are believed to be responsible for
the interaction with fibrin (3-5). tPA is synthesized as a
single-chain polypeptide and can be converted into its more
active two-chain form by plasmin scission of an Arg-Ile bond
situated in the strand connecting kringle 2 with the serine
proteinase domain (6).
Kinetic analysis of the human tPA catalysis was rendered
in a number of papers (7-9) and resulted in several
conclusions concerning its mechanism. The single-chain
enzyme was considered as a poor catalyst when compared
to the double-chain form. It was characterized by K
m
) 1-2
µM toward [Glu
1
]plasminogen and k
cat
) 0.3-0.6 min
-1
(9).
This work is part of the FØTEK program supported by the Danish
Dairy Research Foundation (Danish Dairy Board) and the Danish
Government.
* Corresponding author.
§
University of Aarhus.
|
MD Foods Research and Development Centre.
Present address: Biotechnological Institute, Kogle Alle´ 2, DK-
2970 Hørsholm, Denmark.
1
Abbreviations: [Asp
1
]plasminogen, native form of bovine plas-
minogen with an aspartic acid at the N-terminus; EACA, -aminocaproic
acid; Fb, fibrinogen; [Glu
1
]plasminogen, native form of human plas-
minogen with a glutamic acid at the N-terminus; PAI, plasminogen
activator inhibitor; Pg, plasminogen; Pn, plasmin; tPA, tissue-type
plasminogen activator; uPA, urokinase plasminogen activator.
12631Biochemistry 1998, 37, 12631-12639
S0006-2960(98)00669-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/19/1998

At the same time, the double-chain tPA had a considerably
higher affinity to the substrate K
m
) 0.1-0.5 µM as well as
higher ability for plasminogen transformation k
cat
) 4-5
min
-1
(8). All catalytic characteristics of the single-chain
tPA improved when fibrin (or fibrinogen fragments) was
added to the medium, showing the K
m
and k
cat
values at the
same level as those of the double-chain enzyme (7, 9). The
double-chain tPA also demonstrated sensitivity to fibrin
which was manifested in 5-10-fold increase of k
cat
but
without any changes in K
m
(8). The existence of an active
ternary complex plasminogen-fibrin-tPA, converting bound
plasminogen to plasmin, was proposed to be more favorable
than the action of a fibrin-tPA complex toward free
plasminogen (9). Previous publications about tPA kinetics
mainly concerned the tPA-catalyzed reaction being in steady
state. This is, in part, due to mathematical difficulties in
the approximation of the complex reaction when the tPA-
catalyzed conversion of plasminogen to plasmin was fol-
lowed by cleavage of a measurable substrate by plasmin.
The human tPA might be an inconvenient object for such
investigations as well. Therefore, application of tPA from
another source, provided with an adapted mathematical
mechanism, could shed some light on “hidden” stages of
the process. An appropriate candidate for this inquiry is
bovine tPA, promising to be useful in several aspects.
We have expressed bovine tPA in the methylotrophic yeast
Pichia pastoris and characterized the product from a
structural and kinetical point of view. Comparison between
human and bovine tPA required standardization of the
experimental conditions, therefore activation of both enzymes
was induced by bovine fibrinogen fragments. The bovine
tPA was characterized by a slower equilibration with the
components of the reaction medium when compared to
human tPA. This allowed a detailed analysis of prestationary
kinetics, impossible under the same conditions for the
catalytic reaction performed with human tPA.
MATERIALS AND METHODS
Chemicals and Reagents. P. pastoris GS115 (his4) (10),
the protease deficient strain SMD1168 (his4, pep4), and the
expression vectors pPIC9K and pHIL-D2 were purchased
from Invitrogen Corp. Super Taq polymerase was from HT
Biotechnology, nucleotide triphosphates were from Pharma-
cia, Chameleon mutagenesis kit was from Stratagene, oligo-
nucleotides were from DNA technology (Science park,
Aarhus, Denmark), and all other enzymes were from New
England Biolabs. PCR was performed in a Hybaid ABA-
CUS thermal cycler. Sequencing was performed either with
a Sequenase kit version 2.0 from United States Biochemical
Corporation or with a dye terminator cycle sequencing kit
from Perkin-Elmer. [
35
S]dATP was from Amersham Inter-
national. Sequencing, ligation, transformation of Escherichia
coli, DNA preparation, PCR, and other DNA modifying
processes were performed according to the manufacturers’
recommendations or standard laboratory procedures. Yeast
media were composed as follows: YPD (1% yeast extract,
2% peptone, 2% dextrose), BMGY (1% yeast extract, 2%
peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB,
1% glycerol), BMMY (1% yeast extract, 2% peptone, 100
mM potassium phosphate pH 6.0, 1.34% YNB, 1% metha-
nol), MM (1.34% YNB, 0.00004% biotin, 1% methanol),
and MD (1.34% YNB, 0.00004% biotin, 1% dextrose).
Yeast extract, peptone, YNB, and casamino acids were from
DIFCO, and G418 was from Life Technologies. Recombi-
nant human tPA (Actilyse) and chromozym tPA (CH
3
-SO
2
-
D-Phe-Gly-Arg-pNA) were from Boehringer Mannheim.
S-2251 (H-
D-Val-Leu-Lys-pNA) was from Chromogenix.
Bovine [Asp
1
]plasminogen and PAI-1 were from American
Diagnostica. PAI-2 was of the low molecular weight form
and generously provided by Dr. I. Lecander (Lund, Sweden).
CNBr-fibrinogen fragments were made by incubation of 100
mg fibrinogen with 130 mg CNBr in 70% formic acid at
room temperature for 16 h. The resulting fibrinogen
fragments were dialyzed against water for removal of low
molecular weight fragments (membrane cut off ) 12-14
kDa) and stored at -80 °C at a concentration of 2.8 mg/
mL. The bovine plasminogen used for zymography was
purified as described in ref 11, and plasminogen depleted
bovine fibrinogen was obtained from Enzyme Research
Laboratories.
Construction of the P. pastoris tPA Expression Vectors.
pHIL-D2/tPA was derived from the P. pastoris integration
vector pHIL-D2. The tPA encoding region, including the
native signal peptide, was inserted into the EcoRI restriction
site in pHIL-D2, yielding pHIL-D2/tPA. Due to the presence
of an internal EcoRI restriction site in the tPA cDNA, this
was performed by PCR on pBtPA4 (12) with the forward
5-ATGATGAGCGCAATGAAG-3 and reverse 5-GGT-
GTCCCTGGTCATGG-3 primers. The EcoRI restriction
site in the resulting amplified tPA encoding region was then
methylated by EcoRI methylase and ligated to EcoRI linker
oligonucleotides 5-GGAATTCC-3. Following digestion by
EcoRI, the amplified fragment was ligated into pHIL-D2.
pPIC9K/tPA was derived from the P. pastoris integration
vector pPIC9K. Using PCR, 5 and 3 NotI restriction sites
were introduced into the bovine tPA cDNA using the plasmid
pBtPA4 as template and the following forward and reverse
primers, respectively, 5-CTCAGGAGAGCGGCCGCATCG-
TAC-3 and 5-GAGGAAAGCGGGCGGCCGCCCTGGG-
3. The resulting PCR product was cloned into the NotI
restriction site in pPIC9K. To generate the native N-terminus
of tPA after cleavage by the signal peptidases (Figure 1),
site specific mutagenesis was performed on pPIC9K/tPA,
using the primer 5-CTCGAGAAAAGAGAGGCTGAAGCT-
TCGTACAAAGTGACCTGCAGAGAT-3. To ensure that
the T4 polymerase replicated the entire plasmid, two non-
mutagenic primers, situated in the Col E1 region (7961-
7980) and HIS4 region (4801-4821) of pPIC9K/tPA, were
included in the mutagenesis reaction. The tPA encoding
region of pPIC9K/tPA was finally sequenced as a control
for PCR-introduced mutations.
Transformation of P. pastoris with pPIC9K/tPA and pHIL-
D2/tPA and Multicopy Colony Selection. P. pastoris strains
GS115 (his4) and SMD1168 (his4, pep4) were transformed
with the expression plasmids using the spheroblasting and
FIGURE 1: Partial amino acid sequence of the pPIC9K/tPA secretion
signal fused to the mature N-terminal of tPA is shown, as well as
the expected two endoproteolytic split sites (KEX2, STE13).
12632 Biochemistry, Vol. 37, No. 36, 1998 Johnsen et al.

electroporation techniques. Mut
+
and Mut
s
phenotypes were
determined by screening for fast and slow growth on
methanol-containing plates (MM plates) and, as a control,
evaluating the growth of the same colonies on dextrose-
containing plates (MD plates). Multicopy colonies were
selected by their ability to grow on YPD plates containing
increasing concentrations of G418 (0.25-4 mg/mL). The
transformation, phenotype determination, and selection were
done essentially as described by the supplier.
Fermentation of pPIC9K/tPA Transformed P. pastoris
Strains. Cells were restreaked from freeze cultures on YPD
or MD plates and a single colony was used for inoculation
of 10 mL of BMGY medium. Cells were grown to log
phase, and a suitable volume was used to inoculate 100 mL
of BMGY medium, after which cells were grown until an
OD
600
of 2-6 was reached. Subsequently, the cells were
centrifuged at 1000g at room temperature, the BMGY
medium was discarded, and the cells were resuspended into
200 mL of BMMY medium (with or without 1% casamino
acids) in 2 L baffled shake flasks to an OD
600
) 1. Cells
were grown aerobically (shake flask covered with gaze) and
supplemented with 1% methanol every 24 h. Cells were
removed by centrifugation, and the supernatant was stored
at -80 °C. Cells were always grown in a rotary shaker at
30 °C, 300 rpm. When the expression levels of different
strains were compared, fermentation was performed in a 12
mL reagent glass essentially as described above.
Purification of tPA. All steps were carried out at 4 °C.
Two hundred milliliters of the supernatant was thawed,
centrifuged (10 min, 10000g), dialyzed against 20 mM NaH
2
-
PO
4
, pH 7.0, 100 mM NaCl, 0.05% Tween 80, and applied
to a column of 40 mL of lysine-Sepharose (prepared by
reaction of CNBr-activated Sepharose 4B with lysine). After
the column was loaded, it was washed with 400 mL of 20
mM NaH
2
PO
4
, 200 mM NaCl, and 0.05% Tween 80 and
eluted with the same buffer containing 200 mM
L-arginine.
Fractions were screened for tPA activity, pooled, and
concentrated on an Amicon-30 membrane until A
280
was
approximately 1. The purified protein was stored at -80
°C for later use.
tPA ActiVity Assay and Zymography. tPA activity in
international units (U) was determined by use of the
chromogenic substrate chromozym t-PA according to the
assay described by the manufacturer. The assay was
performed in a total volume of 0.2 mL at 37 °C in microtiter
plates by the addition of the tPA sample to 0.1 M Tris pH
8.5, 0.15% Tween 80, and 0.4 mM chromozym t-PA. The
reaction was followed at 405 nm over a period of1hina
thermostatically controlled Bio-Tek EL 340 BioKinetics
Reader (Bio-Tek Instruments Inc., Winooski, VT). The
amount of U/mL was calculated as follows: (measured
absorbance × min
-1
× mL
-1
tPA sample) × (1 cm/ 0.6 cm,
correction for light path in microtiter wells) × (1/9.75, U
conversion factor). The zymography was performed as
described in ref 13.
Coupled Peptidyl Anilide Plasminogen ActiVation Assay.
The plasminogen activation potential of tPA was evaluated
in a coupled peptidyl anilide assay, where the formation of
plasmin was measured by its hydrolysis of the chromogenic
substrate S-2251. The plasminogen activation reaction was
performed in a total of 0.2 mL containing 0.1 M Tris pH
7.4, 0.02% Tween 80, 0.03-1.08 µM plasminogen, (8.4
µg fibrinogen fragments, and 0.5 mM S-2251. The reaction
was initiated by addition of bovine tPA to the final
concentration of 0.01 µM in the absence of fibrinogen
fragments or 0.001 µM tPA in their presence. The concen-
tration of human tPA was 3.3 × 10
-4
µM in both cases.
The reactions were carried out at 37 °C in microtiter plates
and were followed at 405 nm over a period of1hinthe
same spectrometer as the tPA activity assay. For each assay,
at least two independent experiments were made, with double
determination in each experiment.
Mass Spectroscopy and Amino Acid Sequence Analysis.
Automated Edman degradation was carried out on an ABI
477A/120A protein sequencer (Applied Biosystems) using
standard programs. Mass spectra were acquired using a
matrix-assisted laser desorption ionization (MALDI) mass
spectrometer (Bruker BIFLEX, Bruker-Franzen, Bremen,
Germany) equipped with a nitrogen ultraviolet laser at 337
nm. Samples (2 µL) dissolved in 0.1% trifluoroacetic acid
were mixed with 2 µL R-cyano-4-hydroxycinnamic acid (15
g/L).
Kinetic Analysis of the Coupled Peptidyl Anilide Plasmi-
nogen ActiVation Assay. Three kinetic models were designed
in order to fit the experimental data. The basic model 1
implies the existence of two reactions: (i) transformation
of plasminogen to plasmin catalyzed by tPA, and (ii)
utilization of a chromogenic substrate by plasmin. A new
method of linearization of the initial coordinates (product
versus time) is proposed. A more complex scheme, de-
scribed in model 2, considers the existence of a prestationary
step before the above reactions which distorts linearity of
the plot in its initial part. Model 3 describes the behavior
of the system in the presence of fibrinogen fragments.
Model 1. The dependence of tPA activity on the plasmi-
nogen concentration was investigated in the coupled reaction
assay according to the following schemes
where Pg represents plasminogen, Pn represents plasmin, K
1
and K
2
are the Michaelis constants (K
m
) of the corresponding
enzymes, and k
1
and k
2
are catalytic constants (k
cat
). The
process was followed by conversion of the substrate S-2251
(S) to the colored product (P).
The usual analysis implies transformation of the coordi-
nates ([P] versus time) and plotting of [P] versus t
2
(14).
The curves after transformation are supposed to be linear in
the initial part of the chart where concentrations of both Pg
and S may be considered as constants and are equal to their
initial values [Pg]
0
and [S]
0
. The slopes of these lines are
proportional to Michaelis equation for the first reaction which
allows one to calculate K
1
and k
1
at known K
2
and k
2
. This
method has two major limitations: (i) the linear part can be
manifested only at [S]
0
. K
2
and (ii) any prestationary
kinetics would interfere with the accuracy of determination
(like a lag phase or initial “jump”). Another model has been
developed which includes correction for the decrease in S
concentration (15).
We propose a simple kind of analysis which makes it
possible to use any convenient concentration of S and fit
tPA + Pg S
K
1
tPA-Pg
9
8
k
1
tPA + Pn (S1a)
Pn + S S
K
2
Pn-S
9
8
k
2
Pn + P (S1b)
Two Interconvertible Forms of Bovine tPA Biochemistry, Vol. 37, No. 36, 1998 12633

the curve in the whole range of [P]. Reaction schemes S1a
and S1b can be described by the corresponding system of
differential equations:
The tPA-reaction (S1a) under the chosen conditions was
slow compared to the one catalyzed by Pn (S1b). It satisfied
the requirement [Pg] [Pg]
0
(V
a
const) in the working
time range and allowed a simple integration of eq 1a to [Pn]
)V
a
t. After substitution of [Pn] in eq 1b by V
a
t and
integration, the system was expressed as one equation:
A simple transformation resulted in the linear dependence
of y on t
2
where y ) 2K
2
/k
2
ln(s
0
e
p/K
2
/(s
0
- p)) and y
0
is the error in
determination of the zero point (y
0
0).
The initial coordinates p versus t were transformed to y
versus t
2
using the known values of K
2
) 250 µM and k
2
)
1000 min
-1
(16), as well as the value of s
0
from the
experiment (500 µM). The slope of an individual line was
equal to V
a
at the corresponding [Pg], and the experiments
carried out at different [Pg] gave a set of lines with different
slopes (V
a1
, V
a2
, ...), see Figure 4. The dependence of V
a
on
[Pg] can be fitted according to eq 1a in order to calculate
parameters of the tPA-catalyzed reaction (K
1
and k
1
).
The presence of any prestationary kinetics would disturb
the linearity of the chart y versus t
2
in its initial part. It
might be possible to evaluate the slope of the final linear
component (reached at the stationary conditions) disregarding
the initial shape of the curve if the prestationary step is
relatively quick. One should be careful nominating the linear
part of a curved dependence, as underestimation of the slope
can be quite dramatic due to inappropriate choice.
Model 2. A more sophisticated approach allowed us to
derive some information from prestationary kinetics as well.
From Figure 4A one can see a well defined lag phase in the
reaction with bovine tPA. Its expression increased propor-
tionally to the added Pg, and no linear component could be
found at highest [Pg] in the used time scale. This observation
FIGURE 2: (A) Zymography (lane 1) and SDS-PAGE (lane 2) of
one-chain tPA. (B) SDS-PAGE of one- and two-chain tPA.
Lanes: (1) one-chain tPA unreduced, (2) two-chain tPA unreduced,
(3) one-chain tPA reduced, (4) two-chain tPA reduced.
FIGURE 3: Inhibition of the bovine tPA-mediated plasminogen
activation by PAI-1, PAI-2, and EACA in the presence of fibrinogen
fragments. In the case of inhibition by PAI-1 and PAI-2, 2 pmol
of tPA was incubated at room temperature for 20 min with 9 pmol
PAI-1or PAI-2 before addition to the reaction mixture consisting
of 0.1 M Tris pH 7.4, 0.02% Tween 80, 0.27 µM Plg, 8.4 µg
fibrinogen fragments, and 0.5 mM S-2251 in a total volume of 0.2
mL. EACA was added to the reaction mixture just before initiation
of the reaction. The reaction velocity was calculated as the slope
of the line in a plot of the absorbance versus t
2
.
FIGURE 4: Release of p-nitroaniline after cleavage of the chro-
mogenic plasmin substrate S-2251 in the reaction medium with tPA
and Pg presented in the transformed coordinates y versus t
2
(see
eqs 3 and 6). Solid lines show the best fit according to eq 6 and
model 2. (A) The dependence was obtained for bovine tPA. The
symbols O, 4, 0, ), 3, b, and + correspond to the Pg
concentrations 0.084, 0.19, 0.30, 0.41, 0.52, 0.63, and 0.84 µM,
respectively. (B) The dependence was obtained for human tPA.
The symbols O, 4, 0, ), 3, b, and + correspond to the Pg
concentrations 0.084, 0.19, 0.30, 0.41, 0.52, 0.63, and 0.73 µM,
respectively.
d[Pn]
dt
)V
a
)
k
1
[tPA]
0
[Pg]
K
1
+ [Pg]
(1a)
dp
dt
)
k
2
[Pn](s
0
- p)
K
2
+ (s
0
- p)
(1b)
K
2
ln(s
0
/(s
0
- p)) + p )
1
/
2
k
2
V
a
t
2
(2)
y ) y
0
+V
a
t
2
(3)
12634 Biochemistry, Vol. 37, No. 36, 1998 Johnsen et al.

pointed to a longer time of the prestationary reaction at high
[Pg] when compared to that at low [Pg].
The simplest explanation of the observed phenomenon
implied the existence of two interconvertible tPA conforma-
tions being in equilibrium according to reaction scheme S2
The form tPA
*
cannot bind Pg and is referred to as the
“inactive” enzyme while the “active” tPA is involved in the
reaction with Pg, see reaction schemes S1a and S1b. A lag
phase in the dependence y versus t
2
will be visible when the
initial equilibrium tPA
*
a tPA is shifted to tPA
*
(k
-
. k
+
, [tPA]/[tPA*] , 1) and the conversion between these forms
is relatively slow. The concentration of tPA at the beginning
of the reaction was assumed to be approximately zero in
order to minimize the number of parameters in the equations.
The appearance of Pn in time depends now not only on
the velocity of the tPA reaction itself (V
a
) but also on
equilibration between tPA
*
and tPA:
The rate coefficient k
*
is responsible for the expression of a
lag phase (tPA
*
-tPA equilibration) and depends on the
plasminogen concentration. Increase in [Pg] promotes a shift
in favor of tPA + tPA-Pg which prolongs the equilibration
and decreases the rate coefficient of the prestationary phase
from k
*
) k
+
+ k
-
at [Pg] f 0tok
*
) k
+
at [Pg] f . The
dependence of k
*
on [Pg] has a Michaelis-like nature with
the half saturation parameter equal to K
1
(tPA-Michaelis
constant). Another parameter in eq 4 (V
a
) is a counterpart
of V
a
in eq 1a with the exception of the affinity to
plasminogen reduced here by the factor (1 + k
-
/k
+
).
Integration of eq 4 gives a complex formula for [Pn] as a
function of time:
Substitution of [Pn] in eq 1b and integration provide the
following expression of y as a function of t:
where the notation for y and y
0
is the same as before in eq
3 and V
a
, k
*
are given in eq 4. The chart y versus t
2
has a
tendency to be linear at the sufficiently long time of the
reaction, i.e., y y
0
+V
a
t
2
at t . 1/k
*
. The curves from
Figure 4 were fitted by a nonlinear regression program using
eq 6 to calculate parameters V
a
and k
*
at different [Pg]. Their
values were plotted against [Pg] and analyzed according to
the corresponding formulas in eq 4. The relevant constants
for the tPA reaction (k
+
,k
-
,k
1
,K
1
) were estimated.
Model 3. The presence of fibrinogen fragments in the
reaction medium somewhat complicated the analysis by
adding another component to the system. The lag phase was
already visible both for bovine tPA and human tPA (Figure
5 A,B), and it was expressed at low [Pg] as well as at high
[Pg]. Several models, analogous to reaction scheme S2,
could have caused the appearance of the prestationary stage
with one or another formula for k
*
in eq 6. The scheme
discussed below demands some limits for the value of k
*
which makes it easy to accept or reject this model on the
basis of appropriate or inappropriate fit.
At high concentration of Fb, reaction scheme S3 is reduced
to the following description:
The above model should be supplemented to reaction
schemes S1a and S1b with Fb-tPA written instead of tPA.
The fitting can be performed on the basis of eq 6, where
k
*
) k
+
and the half-maximal value of V
a
is reached at [Pg]
) K
1
. Our attempt to apply reaction scheme S3a for human
tPA (Figure 5B) was quite successful, see the Results. On
the other hand, the fitting curves for bovine tPA (Figure 5A)
showed lower accordance with the experimental data, which
could imply existence of a more complex mechanism than
tPA* {
\
}
k
+
k
-
tPA (S2)
d[Pn]
dt
)V
a
(1 - e
-k
*
t
) (4)
where V
a
)
k
1
[tPA]
0
[Pg]
K
1
(
1 +
k
-
k
+
)
+ [Pg]
, k
*
) k
+
+
k
-
1 +
[Pg]
K
1
[Pn] )V
a
t -
V
a
k
*
+
V
a
k
*
e
-k
*
t
(5)
y ) y
0
+V
a
t
2
-
2V
a
k
*
t +
2V
a
k
*
2
(1 - e
-k
*
t
) (6)
FIGURE 5: Release of p-nitroaniline after cleavage of the chro-
mogenic plasmin substrate S-2251 in the reaction medium with tPA,
Pg, and fibrinogen fragments presented in the transformed coor-
dinates y versus t
2
(see eqs 3 and 6). Solid lines show the best fit
according to eq 6 and the model 3. (A) The dependence was
obtained for bovine tPA. The symbols O, 4, 0, ), and b correspond
to the Pg concentrations 0.05, 0.14, 0.27, 0.41, and 0.68 µM,
respectively. (B) The dependence was obtained for human tPA.
The symbols O, 4, 0, ), 3, and b correspond to the Pg
concentrations 0.05, 0.14, 0.27, 0.41, 0.54, and 0.68 µM, respec-
tively.
tPA* {
\
}
k
+
k
-
tPA + Fb S Fb-tPA (S3)
tPA*
9
8
k
+
Fb-tPA (S3a)
Two Interconvertible Forms of Bovine tPA Biochemistry, Vol. 37, No. 36, 1998 12635

Figures (7)
Citations
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TL;DR: This paper reviews the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced and describes new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.
Abstract: During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

1,960 citations


Journal ArticleDOI
TL;DR: Major advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe’s value and power in commercial and research labs alike.
Abstract: The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

883 citations


Book ChapterDOI
01 Jan 2003
TL;DR: This chapter will review recent research on plasmin and its role in the quality of dairy products and will focus also on other indigenous proteinases, which have not been reviewed thoroughly elsewhere.
Abstract: The presence of indigenous proteolytic activity in milk has been recognized since the work of Babcock and Russel in 1897. Some early researchers attributed this activity to bacterial enzymes, but later work proved conclusively the presence of indigenous proteinases in milk. More recent research has indicated two major categories of indigenous proteolytic enzymes in milk, both originating from the animal’s blood. The major enzyme system contains plasmin, which is produced by activation of its inactive precursor, plasminogen, an event which is under the control of a complex system of inhibitors and activators. The presence of plasmin in milk and its significance to the quality of dairy products has been recognized for many decades (see Bastain and Brown, 1996, for review) and has thus been the subject of much research. However, the other indigenous proteolytic enzymes in milk, which originate from somatic cells, have been studied in detail only during the last decade. Somatic cells, the principal physiological function of which is the defence of the udder against bacterial infection, have lysosomes which contain active proteolytic enzymes, including elastase, collagenase and cathepsins B, D, G, H and L. The acid proteinase originating from somatic cells, cathepsin D, has been studied most thoroughly in milk. However, it is highly likely that the other proteinases known to be present in lysosomes are also present in milk, as has been demonstrated by the recent identification of immunoreactive cathepsin B in milk (Magboul et al., 2001) and observations of the activity of other indigenous thiol proteinases in milk. This chapter will review recent research on plasmin and its role in the quality of dairy products and will focus also on other indigenous proteinases, which have not been reviewed thoroughly elsewhere.

68 citations


Journal ArticleDOI
TL;DR: Extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation and increases enzyme susceptibility to serpins, and DNA is a macromolecular template that both potentiates and inhibits fibrinelysis.
Abstract: The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1–5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α2-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1–1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4–5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α2-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0–20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.

45 citations


Journal ArticleDOI
TL;DR: The study revealed that tetranectin did not interact with the kindred proteins: macrophage-stimulating protein, urokinase-type plasminogen activator and prothrombin, and a kinetic analysis of the tPA-catalysed plAsminogen activation was performed.
Abstract: In the search for new ligands for the plasminogen kringle 4 binding-protein tetranectin, it has been found by ligand blot analysis and ELISA that tetranectin specifically bound to the plasminogen-like hepatocyte growth factor and tissue-type plasminogen activator. The dissociation constants of these complexes were found to be within the same order of magnitude as the one for the plasminogen-tetranectin complex. The study also revealed that tetranectin did not interact with the kindred proteins: macrophage-stimulating protein, urokinase-type plasminogen activator and prothrombin. In order to examine the function of tetranectin, a kinetic analysis of the tPA-catalysed plasminogen activation was performed. The kinetic parameters of the tetranectin-stimulated enhancement of tPA were comparable to fibrinogen fragments, which are so far the best inducer of tPA-catalysed plasminogen activation. The enhanced activation was suggested to be caused by tetranectin's ability to bind and accumulate tPA in an active conformation.

33 citations


References
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Journal ArticleDOI
TL;DR: An approximately 75% pure form of a human Mr approximately 54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose.
Abstract: An approximately 75% pure form of a human Mr approximately 54,000 plasminogen activator inhibitor from conditioned culture fluid of the fibrosarcoma cell line HT-1080 was obtained by a single step of chromatography on concanavalin A-Sepharose. The inhibitor inhibited human urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator, but not plasmin. Rabbit antibodies against this plasminogen activator inhibitor also reacted with a plasminogen activator inhibitor with identical electrophoretic mobility in extracts of human blood platelets, indicating that the HT-1080-inhibitor is of the same type as the inhibitor of blood platelets. As revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin-agarose zymography, incubation of HT-1080-inhibitor with the active form of human u-PA led to the formation of an equimolar sodium dodecyl sulfate-resistant complex between them; in contrast, no complex formation was observed between the inhibitor and the proenzyme form of human u-PA (pro-u-PA). Likewise, using a column of anti-inhibitor antibodies coupled to Sepharose for removal of excess inhibitor and activator-inhibitor complexes, the potential enzymatic activity of pro-u-PA was found to be unaffected by incubation with inhibitor under conditions in which more than 95% of the active u-PA had formed complex with inhibitor.

234 citations


Journal ArticleDOI
TL;DR: Results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment, which was found to be responsible for the binding to lysine-Sepharose or fibrIn.
Abstract: Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment.

90 citations


Journal ArticleDOI
TL;DR: Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles, and was found in acid whey and to a lesser extent in rennet whey.
Abstract: A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino acid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation contained the proteolytically modified midi-plasmin, consisting of kringle 4,5 and the light chain with an apparent molecular weight of 50 k Da in unreduced SDS-PAGE, and resulting in two bands of 30 and 26 k Da after reduction. Some 15% of the amino acid sequence of plasminogen/midi-plasmin isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated from bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 267–278), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593–603). Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen associated with the milk fat globule membrane was very low and reflected the presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85, 80 and 50 k Da in unreduced gels. These hands may represent two plasminogen forms (85 and 80 k Da) and midi-plasmin/midi-plasminogen (50 k Da). The total concentration of plasminogen in bovine milk was estimated to 1.5 μg/mL.

73 citations


Journal ArticleDOI
TL;DR: The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogens to fibrin, which implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the Stability of the Ternary Complex rather than fibrIn binding.
Abstract: The kinetics of activation of both [Glu1]- and [Lys78]Plg(S741C-fluorescein by native (recombinant) tissue-type plasminogen activator and its deletion variants lacking either the finger or kringle-2 domain were measured by fluorescence within fully polymerized fibrin clots. The kinetics conform to the Michaelis-Menten equation at any fixed fibrin concentration so long as the plasminogen concentration is expressed as either the free or fibrin-bound, but not the total. The apparent kcat and Km values both vary systematically with the concentration of fibrin. Competition kinetics disclosed an active site-dependent interaction between t-Pa and [Glu1]Plg(S741C-fluorescein) in the presence, but not the absence, of fibrin. A steady-state template model having the rate equation v/[A]o = kcat(app).[Plg]/(Km(app) + [Plg]) was derived and used to interpret the data. The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogen to fibrin. This implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the stability of the ternary complex rather than fibrin binding.

64 citations


Journal ArticleDOI
Abstract: We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.

55 citations


Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms†" ?

The bovine single-chain tPAmediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. The authors have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The activation of plasminogen has been studied in detail in the human system from where the involved protein components have been identified and characterized. Bovine mastitis is an inflammatory disease of the mammary gland induced by various microorganisms, and a 20-fold increase in tPA activity has been reported in the milk of cows infected with Staphylococcus aureus ( 1 ). The activation of plasminogen by tPA is greatly increased by the Rs2-casein dimer ( 2 ), and in order to study this system in more detail it is necessary to obtain bovine tPA.