The proteolytic activities of specific group of proteases in normo-, micro- and macroalbuminuric HFDa fractions were assayed using 0.1 mM glycyl-prolyl-p-nitroanilide (GP-pNA) and 0.25 mM N-benzoyl-proline-phenylalanine-arginine-p-nitroanilide (Be-PFR-pNA), N-methoxysuccinyl-alanine-alanine-proline-valine-p-nitroanilide (Me-AAPV-pNA), and succinyl-alanine-alanine-proline-phenylalanine p-nitroanilide (Suc-AAPFpNA) chromogenic substrate of chromogenic substrates to establish activity of DPP IV, Kallikreins, proteinase 3, and cathepsins, respectively [23, 33-35].
Beside the relative quantitative amount we checked the activity of some of these proteases by gelatine zymography for metalloproteases (Figure 4) which confirmed a clear increase of gelatinase activity in the normoalbuminuric group and by chromogenic substrates for DPP IV, Kallikreins, cathepsins, and PRTN3 (Figure 5).
Increase of the urinary level of DPP IV associated with microvesicles is correlated with the worsening of DN .
Sun and colleagues  reported an increased excretion of DPP IV associated with microvesicles in type 2 diabetic patients.
August, "Demonstration of glomerular DPP IV activity in kidney diseases," Acta Histochemica, vol.
Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV
. Crit Rev Clin Lab Sci 2003;40:209-94.
Diprotin A as an inhibitor affects the catalytic activity of DPP IV
in the human central nervous system, endocrine system and on the CD-26 of immune system (Maes et al.
Recently, other processed forms of BNP, including BNP(3-32), BNP(4-32), and BNP(5-32), have been detected in plasma from heart failure patients in the presence of protease inhibitors benzamidine (as a trypsin, plasmin, thrombin inhibitor) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (as an inhibitor for serine protease such as DPP IV
) to minimize the effect of protease degradation (15).
The substrate specificity of DPP IV is not that strict, however; cleavage after a penultimate Ser, Gly, Thr, Val, and Leu also has been observed.
ANP (1-28) has a leucine and proANP (79 -98) has a serine in this position, but these are not the preferred amino acids for DPP IV.
The first aim of this study was to ascertain whether BNP (1-32), ANP (1-28), proANP (1-30), and proANP (79-98) are truncated by DPP IV and to determine the kinetic data of the observed cleavages.
Soluble DPP IV was purified from human seminal fluid as described previously (19).