Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 44: 502-508, 1998;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Küllertz, G.
Right arrow Articles by Fischer, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Küllertz, G.
Right arrow Articles by Fischer, G.
Related Collections
Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 1998;44:502-508.)
© 1998 American Association for Clinical Chemistry, Inc.

Enzymes and Protein Markers

Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera

Gerhard Küllertz1,a, Sabine Lüthe2, and Gunter Fischer1

1 Max-Planck Research Unit, Enzymology of Protein Folding, Kurt-Mothes Str. 3, D-06120 Halle/Saale, Germany.

2 Martin-Luther-University Halle-Wittenberg, Institut for Clinical Chemistry and Pathobiochemistry, Madgeburger Str., D-06112 Halle/Saale, Germany.
a Author for correspondence. Fax +49–345 5511972;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P <0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Helper enzymes for protein folding may be involved in many pathological processes thought to comprise diseases known to be caused by defective protein folding (1). The ubiquitously distributed peptidylprolyl cis/trans isomerases (PPIases, EC 5.2.1.8) represent a major class of folding helper enzymes (2)(3)(4)(5).1 Among the three families making up this enzyme class, cyclophilins (Cyps) have been characterized as receptors for the immunosuppressant cyclosporin A, whereas a second PPIase family, the FK506 binding proteins (FKBPs), represents cellular receptors for the immunosuppressive peptidomacrolide FK506 (2)(3)(4).

Numerous Cyps and FKBPs have been detected in human tissues (5)(6)(7)(8)(9)(10)(11) and body fluids (6)(7)(8)(9)(10)(11)(12)(13)(14), all of which exhibit considerable enzymatic activity toward proline-containing oligopeptides. Apart from their role as targets of immunosuppressants, PPIases are associated with a variety of biological functions. PPIases have been isolated as components of steroid hormone complexes (3)(4)(5)(15)(16) and have a putative function in structuring unfolded proteins (3)(17). They are probably able to affect cells in a pleiotropic manner (2)(3), and are also associated with a variety of Ca2 handling mechanisms in mitochondria (18)(19) and the sarcoplasmic reticulum (20). The aim of this study was to assess whether pathological processes are reflected in alterations of PPIase activity in body fluids or tissues. Because of the peculiarities of the reversible cis/trans isomerization preventing simple end-point determination, a semiautomated PPIase assay with high throughput is an indispensable tool to detect such changes.

To date, the concentration of individual PPIases has been determined with enzyme-linked immunosorbent systems (8)(9)(12)(19)(21) or radioactive binding assays directed against any of the immunosuppressants (6)(8)(12).

It is striking that neither investigations on PPIase activity in human blood plasma or serum nor results on the pathophysiological significance of these enzymic activities in blood plasma or serum have been published.

Conventionally, the enzymatic activity of PPIases has been determined with the aid of UV/VIS spectrophotometry with N-succinylated tetrapeptide-4-nitroanilides as standard substrates. This assay is based on monitoring the time course of the chymotryptic cleavage of the 4-nitroanilide bond, which is kinetically coupled to the cis-to-trans isomerization of the peptidylprolyl bond of the substrate. Since the spontaneous cis-to-trans isomerization was found to be too fast for reliable calculation of rate constants at room temperature, the assay is usually carried out at or below 10 °C. A typical half time of the uncatalyzed cis-to-trans isomerization is about 100 s at 10 °C.

The assay has some technical disadvantages that may affect implementation into the routine procedures of medical laboratories. For example, the total run time for monitoring the PPIase activity of just one sample takes about 20 min. Moreover, the quality of the kinetic traces produced under assay conditions is vitally dependent on the rapidity, effectivity, and reproducibility of the mixing procedure used for the reagents. These preconditions are difficult to maintain by manual mixing procedures. To solve these disadvantages, we have developed a semiautomated PPIase assay in a 96-well microtiter plate that permits the reproducible determination of PPIase activities with a high throughput rate in crude biological fluids.

In this report, we describe the evaluation of this assay concerning both reliability and accuracy using human sera as the source of PPIase activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals
Substrates, succinyl-Phe-Pro-Phe-4-nitroanilide, and succinyl-Ala-Xaa-Pro-Phe-4-nitroanilides, with Xaa being Phe, Ala, Lys, Ile, Gln, or Trp, were obtained from Bachem. Succinyl-Ala-Ala-Pro-Phe-OH was synthesized by solid-phase peptide synthesis with chlorotritylchloride resin and kindly provided by M. Schutkowski (University of Halle). Inhibition experiments were carried out with FK506 (Fujisawa) and cyclosporin A (AWD). Bovine {alpha}-chymotrypsin was obtained from E. Merck and was found to contain 58% active enzyme as determined by active-site titration with 4-nitrophenyl acetate (22). Human recombinant Cyp (Cyp18cy; nomenclature of PPIases published in ref. 3) and human recombinant FKBP12 were obtained from Boehringer Mannheim. Other chemicals used in this study were of analytical purity and obtained commercially.

collection of samples
Human blood samples were collected by venipuncture and after clotting, the serum was separated by centrifugation at 700g for 15 min. Heparin, EDTA, or citrate plasma was prepared (Sarstedt) with the corresponding anticoagulants. PPIase activities in sera were determined within 12 h after venipuncture. Other investigations involving serum or plasma samples were carried out immediately after centrifugation. Samples were collected from 151 healthy adult volunteers (83 females, 69 males) and 47 unselected patients (24 females, 23 males). The investigated samples were from individuals with heart diseases [cardiac infarction (n = 4) and other (n = 4)], kidney diseases (n = 4), acute and chronic liver diseases [acute hepatitis (n = 4), chronic hepatitis (n = 6), cirrhosis (n = 5), and cholestatic liver diseases (n = 4)], insulin-dependent diabetes (n = 3), chronic hemodialysis (n = 6), cancer (n = 4), and chronic pancreatitis (n = 3).

laboratory measurements
In some cases the serum laboratory values of the patients were heavily pathological as recognized by means of percentile points (5%, 50%, and 95%): creatinine (56.8, 76.5, 229 µmol/L), urea (1.8, 5.9, 16.9 mmol/L), uric acid (181, 351, 586 µmol/L), bilirubin total (6, 13, 26 µmol/L), aspartate aminotransferase (AST) (252, 424, 849 U/L), alanine aminotransferase (ALT) (228, 432, 1610 U/L), {gamma}-glutamyltransferase (GGT) (169, 815, 4552 U/L), alkaline phosphatase (AP) (1.2, 2.4, 9.3 U/L), {alpha}-amylase (0.9, 1.9, 6.2 U/L), lipase (25.2, 110, 1949 U/L), lactate dehydrogenase (LDH) (2.6, 5.7, 16.2 U/L), cholesterol (1.8, 5.0, 10.2 mmol/L), triglycerides (0.8, 1.4, 4.0 mmol/L), and C-reactive protein (4.9, 19.1, 1222 mg/L). For all measurements commercially available kits were used.

determination of ppiase activity
PPIase activity was measured spectrophotometrically at 390 nm at 5 °C, with bovine {alpha}-chymotrypsin as an isomer-specific protease. The time course of the hydrolysis of the cis conformer in the absence and presence of PPIase was used to calculate enzyme activity as

(1)
in arbitrary units (K) with kobs as the first-order rate constant of the PPIase-catalyzed reaction and ko as the uncatalyzed cis-to-trans interconversion (3). The measurements were performed either conventionally with a diode array spectrophotometer (HP8452A, Hewlett Packard) and the equipment previously described (2)(17), or applying the semiautomated method with a microplate reader (MR7000, Dynatech). The microplate assay was used as a routine assay to measure the PPIase activity in serum of patients. The assay itself was carried out in a cold-storage device at 6 °C. The precision, in terms of CV of the absorbance of twelve repetitive recordings (390 nm, 150 µL of buffered solutions of 4-nitroaniline per well), was ±0.9% up to an absorbance of 0.9 and increased up to ±1.5% at an absorbance of 2. Therefore, we used 17 nmol of substrate per cavity to give an absorbance of approximately 0.7 after completion of the reaction. This substrate concentration takes into account the contribution of the intrinsic absorbance of the added serum to the total absorbance, which has to fall below a value of 0.9 at 390 nm.

When the conventional method was used, the sample cell (total volume 1.257 mL) contained 1.2 mL of 35 mmol/L HEPES buffer (pH 7.8), 600 µg of chymotrypsin, and 50 µL of serum. Instead of serum, the control contained 50 µL of water. The mixture was incubated at 5 °C and the reaction was started by adding 7 µL of substrate stock solution (10–20 mg of the corresponding substrate in 1 mL of dimethyl sulfoxide). The liberation of 4-nitroaniline was observed at 390 nm for at least 15 min with a sampling rate of 120 data points per min.

In the 96-well nonsterile (flat bottom) microtiter plates (Bibby Sterilin) a total volume of 137 µL of solution per cavity, consisting of 3–12 µL of serum, 50 µL of substrate solution (125 mg/L succinyl-Phe-Pro-Phe-4-nitroanilide in 35 mmol/L HEPES buffer, pH 7.8), and 80 µL of chymotrypsin solution (1 g/L chymotrypsin in 35 mmol/L HEPES buffer, pH 7.8), was used. The reaction was started by adding the substrate solution with the microplate reader dispenser, followed by rapid and intense mixing (PLAT-MIX, Garching Innovation) of the reaction mixture in each cavity. The first data point of the reaction was recorded 20 s after reaction start and the sampling rate was 10 data points per 90 s. Collection was finished after storage of 70 data points for each well. Typical time courses of catalyzed and uncatalyzed coupled assays are shown in Fig. 2 .



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Absorbance plot of the time course of the PPIase assay in sample cells of a 96-well microtiter plate at 390 nm.

As the standard test, the chymotrypsin-coupled monitoring of the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide was used at 5 °C. The figure on the left (a–e) represents the effect of decreasing chymotrypsin concentrations (570, 170, 113, 75, 50, and 33 mg/L). In the figure on the right (a–d) the effect of gradually decreasing serum (pooled sample with a PPIase activity of 87 K) concentrations (16.55%, 2.27%, 0.48%, and 0% in the sample cell) is shown.

upper limit of enzyme activity
Usually, first-order rate constants (k) in isomer-specific proteolysis are calculated with the equation

(2)
with A{infty} as the final absorbance of the chromogenic product, At as the absorbance at time t, and Ao as the amplitude of the fast phase of isomer-specific proteolysis (Fig. 1 ). For the coupled PPIase assay, kobsUL represents the upper limit of first-order rate constants that can be determined with the used assay system. It can be calculated by expressing A{infty} - At in terms of Ppre (the precision of the photometer), by substitution of A{infty} - Ao by (A{infty} CCis%)/100 (with CCis% as the percentage of cis conformer), and by expressing t in terms of tlag (Dp Drate) to give

(3)
where Drate is the data sampling rate, Dp is the minimal number of data points required for precise calculation of k, and tlag is the lag time between reaction start and reliable recording at the first data point of cis-to-trans isomerization. Combining Eqs. 1Up , and 3Up gives the upper limit of arbitrary units (KUL) that can be measured in the sample cell:

(4)
By using Eq. 4Up and the values specific for the microplate reader used (A{infty} = 0.7, Ppre = 0.008, tlag = 20 s, Drate = 9.5 s, and Dp = 10) in conjunction with the substrate-specific constants as ko and CCis% (Table 1 ), substrates can be ranked according to their applicability in the assay.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Schematic drawing of a time course of isomer-specific proteolysis of a chromogenic tetrapeptide 4-nitroanilide as monitored by 4-nitroaniline release.

to, reaction start; A{infty}, final absorbance; Ao, slow-phase extrapolation to to; tlag, lag time of monitoring (mixing period, cleavage of trans isomers).


View this table:
[in this window]
[in a new window]
  		Table 1. Assay-related characteristics defining PPIase substrates

statistical analysis
Statistical calculations with the software CSS:STATISTICA (StatSoft) were used to analyze the relation between laboratory variables and PPIase activity, and to determine the standard deviation of kinetic constants.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
choice of substrate
We assayed PPIase activities in a pooled serum probe for six tetrapeptides and a tripeptide substrate. The highest PPIase activity was detected with succinyl-Ala-Gln-Pro-Phe-4-nitroanilide. Because of the preference of succinyl-Ala-Ala-Pro-Phe-4-nitroanilide for human Cyp18 (23) and succinyl-Ala-Leu-Pro-Phe-4-nitroanilide for human FKBP12 (23), the specificity found for serum on the PPIase activity indicated the presence of still uncharacterized PPIases in the serum pool. However, the selection of an optimal substrate requires additional considerations regarding the rate constant ko (a low value is preferable) and the percentage of cis isomer (which should be high). Both boundary conditions contribute considerably to the upper limit of arbitrary constants KUL (Table 1Up , Eq. 3Up ), which quantitatively describe the suitability of substrates for the semiautomated assay. The substrate with the broadest applicability in the microtiter plate-based assay was succinyl-Phe-Pro-Phe-4-nitroanilide. Because this substrate is also a sensitive indicator of serum PPIase activity, it was used as standard substrate in our routine measurements.

substrate concentration
An oligopeptide containing a prolyl bond, where the cis conformer is used as a substrate in any protease-coupled PPIase assay, consists of a mixture of two conformers, cis and trans, in solution (2)(22)(23)(24)(25)(26). Dependent on the nature of the amino acid flanking the proline residue of substrates, the percentage of cis conformer in aqueous solutions is in the range of 5–35% (3). Therefore, only 5–35% of the total absorbance signal can be used for assaying PPIases by a protease-coupled reaction. Because of the peculiarities of the coupled assay, the remaining part contributes to the unwanted background absorbance attributed to 4-nitroaniline at 390 nm (15)(24)(25). For a given peptide, the ratio of the conformers can be influenced by the solvent as LiCl-tetrahydrofuran (24), aqueous detergents (23), or the buffer conditions. In dimethyl sulfoxide, the substrate succinyl-Phe-Pro-Phe-4-nitroanilide has 28% cis conformer (23), whereas in aqueous buffer solution it rises to a value of 35% (Table 1Up ).

A condition for quantifying PPIase activities from time courses of coupled reactions by applying the first-order rate law is a relation between substrate concentration [S] and Michaelis constant (KM) of [S] KM. The presence of PPIases with high affinity for a substrate (KM < [S]) is usually indicated by deviations of the experimental time traces from strict first-order kinetics in the early phases of the PPIase-catalyzed reaction. However, no such evidence was observed for all combinations of substrates and serum samples used here. Purified PPIases, as Cyp18 or FKBP12, have also been investigated in this respect (2)(23)(25). They were found to fit exactly the kinetic law discussed above when using substrate concentrations <120 µmol/L. Although it is known that blood sera of healthy subjects contain secretory Cyp23 (12) as a major PPIase, and that the concentration of FKBP12 increases in sera of renal transplant patients (21), pathological sera might contain several additional PPIases with KM values >= [S]. Numerous human PPIases of all three families have been discovered (6)(7)(8)(9)(10)(12)(13)(14)(21)(27)(28)(29)(30), but there is only scant information on substrate specificities or KM values. Moreover, the exact PPIase composition of patient sera is still unknown.

To consider the influence of the proteolytic peptidylprolyl fragment, which is produced in large amounts in the fast phase of the coupled reaction, we investigated the influence of succinyl-Ala-Ala-Pro-Phe-OH on the PPIase activity of a mixture of 10 sera and in a separate experiment with purified Cyp18 and FKBP12. Because of its proline content, it has, in principle, a potential to compete with the uncleaved chromogenic cis substrate for the active site of the PPIase. Because no effect on the PPIase activity was found after addition of up to 1 mmol/L succinyl-Ala-Ala-Pro-Phe-OH (not shown), the used substrate concentration, <200 µmol/L, appears to be suitable for a kinetically unperturbed PPIase assay in human blood sera.

concentration of isomer-specific protease
The helper protease must fit two essentials: first, the protease should exhibit a high degree of isomer specificity toward the substrate of choice. In addition, the hydrolysis of the trans isomer should be fast enough to ensure that the portion of the substrate with the peptidylprolyl bond in the trans conformation will be completely hydrolyzed within the mixing time tlag of the reagents (fast phase of the reaction, Fig. 1Up ). The kinetics of the remaining part of the reaction then entirely corresponds to the cis-to-trans isomerization of the peptidylprolyl bond (slow phase of the reaction). Chymotrypsin is the protease used most frequently in PPIase assays, followed by subtilisin or thrombin (3)(31). We investigated the influence of the chymotrypsin concentration on the kinetic pattern of 4-nitroaniline release from succinyl-Phe-Pro-Phe-4-nitroanilide in the presence of 16.6% of a pooled serum (Figs. 2Up and 3). Only at chymotrypsin concentrations >400 mg/L was a ratio of amplitudes of two kinetic phases found, which corresponds to the actual cis-to-trans ratio of the substrate. This reveals that the assay conditions are appropriate for measuring unperturbed kinetics of cis-to-trans isomerization in serum samples.

Second, the protease should not compromise the serum PPIases by digestion during the operation time of the assay. We measured the PPIase activity in serum after preincubation of eight different serum samples with chymotrypsin at 5 °C. Even at the high concentration of chymotrypsin (500 mg/L) necessary for the assay, there is no indication of a loss of PPIase activity after a preincubation time of 120 min.

precision of the first-order rate constant
The rate constants of the catalyzed first-order cis-to-trans interconversion (kobs) were calculated from the time course of the absorbance (3). To determine the precision of the calculated rate constants, serum in increasing amounts was applied (Figs. 2Up and 4 ). At a rate constant <0.03 s-1, when the half time of the reaction is higher than the mixing time, the first-order reaction is obviously slow enough for precise determination with the microplate reader technique (Fig. 4 ). This value corresponds to the upper limit calculated by Eq. 3Up with the following values: CCis% = 28%, A{infty} = 0.7, Ppre = 0.008, tlag = 20 s, Drate = 9.5 s, Dp = 10.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Dependence of first-order rate constants kobs on serum concentration in the assay.

With the data points of eight determinations of kobs, a linear regression [r = 1, b(0) = 0.501 ± 0.002, b(1) = 0.2070 ± 0.0004] was calculated. Inset: standard deviation of the of kobs values.

precision of the rate factor K
Calculations were performed according to Eq. 1Up , which describes the PPIase activity in the sample cell as arbitrary units. By applying the dilution factor for the serum sample, the magnitude of K immediately reports the actual capacity of undiluted serum to accelerate the rate of the cis-to-trans isomerization of the reference peptide. The precision of K depends on the error in the determination of ko and kobs (Eq. 1Up ). Whereas the error in ko is independent of the serum sample, the error in K is likely to be dependent on the absolute magnitude of kobs. The highest precision of K is found in the range of kobs between 0.01 s-1 and 0.025 s-1. To get a maximal accuracy of K, the dilution factor of serum should be adjusted to fit this optimal range. The first indication of a compromised kobs can be obtained by evaluating the ratio of the kinetic amplitudes, which must be in the range of 34% ± 3%; other values signalize lack of precision of the calculated kobs value (Fig. 3 ).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Influence of chymotrypsin concentration on the rate constant and the amplitude calculated by applying Eq. 2Up to the time trace: ... ... , percentage of the slow phase of the isomer specific proteolysis; ——, calculated first-order rate constant kobs.

Error bars represent the standard deviations of three determinations.

stability of ppiase activity in stored sera
Peptidylprolyl cis/trans isomerase activity in normal (n = 10) and pathological (n = 10) human sera was found to be stable for at least 2 months when the sera were stored at -80 °C and for at least 96 h at 4 °C. The activity continually decreased to about 50% of the original value within 12 h at room temperature. After blood clotting and centrifugation, the sera should be separated immediately. Up to a twofold increase of PPIase activity was observed in serum samples not separated from blood clot after 8 h. Obviously, PPIases localized in erythrocytes (9)(10) were released. A similar increase was observed for LDH and electrolytes as well (32).

ppiase activities in normal and pathological sera
Because no significant differences between serum and heparin plasma were found, serum was used.

The PPIase activity patterns of the 151 healthy adult volunteers and the 47 patients (Fig. 5 ) were shown to possess sex-specific differences. The 5th, 50th, and 95th percentiles of the PPIase activities calculated for healthy individuals (83 females and 69 males) were 14, 30, and 48, and 17, 36, and 55 K, respectively. With the nonparametric Mann–Whitney rank-sum test, this sex-specific difference was significant, with a P value <0.0035.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 5. Frequency histogram for PPIase activity in sera of 151 healthy donors (69 males, 83 females) and in sera of 47 patients.

In both a healthy population control group and patients, men and women showed the same age distribution, with 19.1 and 59.2 years as values of the 5th and 95th percentiles respectively. A statistically significant correlation between PPIase activity and age was not detected.

Healthy donors revealed significantly higher values (P = <0.0001, Mann–Whitney rank-sum test) compared with the 47 serum samples from different patients with pathological laboratory variables (Fig. 5Up ). Remarkably, all correlations (Pearson product–moment correlation) between any of these laboratory variables and PPIase activity were inverse (data not shown) but not statistically significant.

It was surprising that AP or GGT activities did not correlate with PPIase activity because liver cells contain relatively high amounts of PPIase (8). Therefore we proved the presence of an effector. With succinyl-Phe-Pro-Phe-4-nitroanilide as substrate and sera from patients with high and low PPIase activity, the correlation between serum concentration in the assay and PPIase activity was investigated. No significant deviation from the expected linear relation between activity and concentration was detected between 0.5% and 15% (by vol) of serum in the assay. Mixtures of sera with high and low activity showed the calculated PPIase activity values in all cases. Therefore, we are able to exclude an endogenous effector in serum that could account for variability in serum activity (Fig. 4Up ).

It remains unanswered whether the internalization of serum PPIases into T lymphocytes, as described for Cyp23 (33), or other unknown effects are responsible for the lower PPIase activity in serum. Because PPIases bind cyclosporin A and FK506, thereby influencing distribution of both immunosuppressants in the body (5), knowledge about the variability of the PPIase concentration should be of pharmacological interest.

In summary, the above results constitute proof of suitability of a 96-well microtiter plate PPIase assay for determining PPIase activities in sera. It is reliable in terms of sensitivity and precision. First results concerning PPIase activity in serum of healthy donors and patients provide an impression of how large the rate of cis-to-trans isomerization of a reference peptide in this body fluid really is.


   Acknowledgments
 
This work was supported by the Deutsche Forschungsgesellschaft (Fi 455/3–3), the Fond der Chemischen Industrie, and the Boehringer Ingelheim Stiftung. We thank Michelle Tolliday for critical reading of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: PPIase, peptidylprolyl cis/trans isomerase; Cyp, cyclophilin; FKBP, FK506 binding protein; GGT, {gamma}-glutamyltransferase; AP, alkaline phosphatase; and LDH, lactate dehydrogenase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Welch WJ, Brown CR. Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1996;2:109-115.
  2. Fischer G, Wittman-Liebold B, Lang K, Kiefhaber T, Schmid FX. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 1989;337:476-478. [Medline] [Order article via Infotrieve]
  3. Fischer G. Peptidyl-prolyl cis/trans isomerases and their effectors. Angew Chem Int Ed Engl 1994;33:1415-1436.
  4. Galat A, Metcalfe SM. Peptidylproline cis/trans isomerases. Progr Biophys Mol Biol 1995;63:67-118. [Web of Science][Medline] [Order article via Infotrieve]
  5. Lawen A. Biosynthesis and mechanism of action of cyclosporins. Progr Med Chem 1996;33:53-97. [Medline] [Order article via Infotrieve]
  6. Koletsky AJ, Harding MW, Handschumacher RE. Cyclophilin: distribution and variant properties in normal and neoplastic tissues. J Immunol 1986;137:1054-1059. [Abstract]
  7. Schneider H, Charara N, Schmitz R, Wehrli S, Mikol V, Zurini MGM, et al. Human cyclophilin C: primary structure, tissue distribution, and determination of binding specificity for cyclosporins. Biochemistry 1994;33:8218-8224. [Medline] [Order article via Infotrieve]
  8. Marks WH, Harding MW, Handschumacher R, Marks C, Lorber MI. The immunochemical distribution of cyclophilin in normal mammalian tissues. Transplantation 1991;52:340-345. [Web of Science][Medline] [Order article via Infotrieve]
  9. Ryffel B, Woerly G, Greiner B, Haendler B, Mihatsch MJ, Foxwell BMJ. Distribution of the cyclosporine binding protein cyclophilin in human tissues. J Immunol 1991;72:399-404.
  10. Küllertz G, Meyer S, Fischer G. Differentation by preparative continuous free flow–isoelectric focusing of cyclosporin A inhibitable peptidyl-prolyl cis/trans isomerase of human erythrocytes. Electrophoresis 1994;15:960-967. [Web of Science][Medline] [Order article via Infotrieve]
  11. Kay JE. Structure–function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases. Biochem J 1996;314:361-382.
  12. Allain F, Boutillon C, Mariller C, Spik G. Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies. J Immunol Methods 1995;178:113-120. [Web of Science][Medline] [Order article via Infotrieve]
  13. Billich A, Winkler G, Aschauer H, Rot A, Peichl P. Presence of cyclophilin in synovial fluids of patients with rheumatoid arthritis. J Exp Med 1997;185:975-980. [Abstract/Free Full Text]
  14. Lu KP, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 1996;380:544-547. [Medline] [Order article via Infotrieve]
  15. Kieffer LJ, Seng TW, Li W, Osterman DG, Handschumacher RE, Bayney RM. Cyclophilin-40, a protein with homology to the P59 component of the steroid receptor complex. Cloning of the cDNA and further characterization. J Biol Chem 1993;268:12303-12331. [Abstract/Free Full Text]
  16. Yem AW, Tomasselli AG, Heinrikson RL, Zurcher-Neely H, Ruff VA, Johnson RA, Deibel MR, Jr. The Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13. Biol Chem 1992;267:2868-2871. [Abstract/Free Full Text]
  17. Lang K, Schmid FX, Fischer G. Catalysis of protein folding by prolyl isomerase. Nature 1987;329:268-270. [Medline] [Order article via Infotrieve]
  18. Connern CP, Halestrap AP. Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to [Ca2+]. Biochemistry 1996;35:8172-8180. [Medline] [Order article via Infotrieve]
  19. Bram RJ, Crabtree GR. Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. Nature 1994;371:355-358. [Medline] [Order article via Infotrieve]
  20. Timerman AP, Onoue H, Xin HB, Barg S, Copello J, Widerrrecht G, Fleischer S. Selctive binding of FKBP12.6 by the cardiac ryanodine receptor. J Biol Chem 1996;271:20385-20391. [Abstract/Free Full Text]
  21. Shirakata Y, Kobayashi M, Ohtsuka K, Sugano M, Terajima H, Ikai I, et al. Inhibitory effect of plasma FKBP12 on immunosuppresive activity of FK506. Transplantation 1995;60:1582-1587. [Web of Science][Medline] [Order article via Infotrieve]
  22. Schutkowski M, Wöllner S, Fischer G. Inhibition of peptidyl-prolyl cis/trans isomerase activity by substrate analog structures: thioxo tetrapeptide-4-nitroanilides. Biochemistry 1995;34:13016-13026. [Medline] [Order article via Infotrieve]
  23. Harrison RK, Stein RL. Substrate specificities of the peptidyl-prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes. Biochemistry 1990;29:3813-3816. [Medline] [Order article via Infotrieve]
  24. Kofron JL, Kuzmic P, Kishore V, Gemmecker G, Fesik SW, Rich DH. Lithium chloride perturbation of cis-trans peptide bond equilibria—effect on conformational equilibria in cyclosporin-A and on time-dependent inhibition of cyclophilin. J Am Chem Soc 1992;114:2670-2675.
  25. Fischer G, Bang H, Mech C. Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides. Biomed Biochim Acta 1984;43:1101-1111. [Web of Science][Medline] [Order article via Infotrieve]
  26. Kramer ML, Fischer G. FKBP-like catalysis of peptidyl-prolyl bond isomerization by micelles and membranes. Biopolymers 1997;42:49-60.
  27. Bergsma DJ, Eder C, Gross M, Kersten H, Sylvester D, Appelbaum E, et al. The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms. Biol Chem 1991;266:23204-23214. [Abstract/Free Full Text]
  28. Anderson SK, Gallinger S, Roder J, Frey J, Young HA, Ortaldo JR. A cyclophilin-related protein involved in the function of natural killer cells. Proc Natl Acad Sci U S A 1993;90:542-546. [Abstract/Free Full Text]
  29. Jin YJ, Albers MW, Lane WS, Bierer BE, Schreiber SL, Burakoff SJ. Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13. Proc Natl Acad Sci U S A 1991;88:6677-6681. [Abstract/Free Full Text]
  30. Wiederrecht G, Martin MM, Sigal NH, Siekierka JJ. Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein. Biochem Biophys Res Commun 1992;185:298-303. [Web of Science][Medline] [Order article via Infotrieve]
  31. Stoller G, Tradler T, Rucknagel KP, Rahfeld JU, Fischer G. An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity. FEBS Let 1996;384:117-122.
  32. Thomas L. Labor und Diagnose, 4nd ed. Marburg, Germany: Medizinische Verlagsgesellschaft, 1992:1936pp.
  33. Allain F, Denys A, Spik G. Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface. Biochem J 1996;317:565-570.



The following articles in journals at HighWire Press have cited this article:


Home page
 J Biomol ScreenHome page
T. Mori, S. Itami, T. Yanagi, Y. Tatara, M. Takamiya, and T. Uchida
Use of a Real-Time Fluorescence Monitoring System for High-Throughput Screening for Prolyl Isomerase Inhibitors
J Biomol Screen, April 1, 2009; 14(4): 419 - 424.
[Abstract] [PDF]

Home page
 Biol. Reprod.Home page
A. N. Santos, S. Körber, G. Küllertz, G. Fischer, and B. Fischer
Oxygen Stress Increases Prolyl cis/trans Isomerase Activity and Expression of Cyclophilin 18 in Rabbit Blastocysts
Biol Reprod, January 1, 2000; 62(1): 1 - 7.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
M. Metzner, G. Stoller, K. P. Rucknagel, K. P. Lu, G. Fischer, M. Luckner, and G. Kullertz
Functional Replacement of the Essential ESS1 in Yeast by the Plant Parvulin DlPar13
J. Biol. Chem., April 20, 2001; 276(17): 13524 - 13529.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Küllertz, G.
Right arrow Articles by Fischer, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Küllertz, G.
Right arrow Articles by Fischer, G.
Related Collections
Right arrow Proteomics and Protein Markers

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS