Ala-Pro-cresyl violet, a synthetic fluorogenic substrate for the analysis of kinetic parameters of dipeptidyl peptidase IV (CD26) in individual living rat hepatocytes.

نویسندگان

  • C J Van Noorden
  • E Boonacker
  • E R Bissell
  • A J Meijer
  • J van Marle
  • R E Smith
چکیده

A new type of fluorogenic substrates for proteases based on the leaving group cresyl violet has been synthesized. Cresyl violet is not fluorescent when amino acids or peptide groups are attached but becomes highly fluorescent after proteolytic liberation. Its fluorescence shows linearity with concentration and barely any fading. The properties of Ala-Pro-cresyl violet as substrate for dipeptidyl peptidase IV (DPPIV) (CD26) for localization and quantification of its activity in individual freshly isolated living rat hepatocytes were investigated using confocal microscopy, image analysis, and flow cytometry. DPPIV activity was localized exclusively in patches at plasma membranes likely being bile canalicular domains. Activity was analyzed quantitatively in individual cells by capturing series of images in time. Production of fluorescence was analyzed on the basis of the series of digital images and it appeared to be nonlinear with time. By calculation of the initial velocity at time zero, activity of DPPIV per individual hepatocyte was calculated. Cresyl violet-dependent fluorescence appeared in a similar way when cells were analyzed by flow cytometry. A dipeptide phosphonate inhibitor inhibited production of fluorescence competitively with a Ki of 7 microM. K(m) values in individual hepatocytes varied in the range of 6-22 microM depending on the individual rat from which the hepatocytes were obtained, whereas the Vmax varied in the range of 4-16 nU. K(m) and Vmax values per individual rat were inversely correlated indicating posttranslational regulation of the kinetic parameters of DPPIV. This relationship was lost when membrane fractions of the same hepatocyte suspensions were analyzed. It is concluded that cresyl violet-based protease substrates are the compounds of choice to localize and quantify protease activity in living cells and tissues.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Rapid assay to detect possible natural substrates of proteases in living cells.

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional o...

متن کامل

CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry.

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was spec...

متن کامل

Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).

The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme,...

متن کامل

Activity of Dipeptidyl Peptidase-IV/CD26 and Aminopeptidase N/CD13 in Secretome of Mesenchymal Stem Cells after Treatment with LPS and PMA

Background: Emerging evidence suggests that secretome of mesenchymal stem cells has many anti-inflammatory and regenerative properties, which makes it a suitable candidate for the treatment of autoimmune and degenerative diseases. Dipeptidyl Peptidase-IV (DPP-IV)/CD26 and Aminopeptidase N (APN)/CD13 are ubiquitous ecto-enzymes which can digest various substrates including some ...

متن کامل

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics.

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-psi[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Analytical biochemistry

دوره 252 1  شماره 

صفحات  -

تاریخ انتشار 1997