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The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.

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A proline-rich cytokine from neurosecretory granules of bovine neurohypophysis, 15 amino acids containing PRP-1 (Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr), had been demonstrated as a unique regulator of activity of neurons, strong antibacterial agent, and mediator of the hypothalamus-neurohypophysis-bone marrow-thymus axis, which participates in hematopoietic stem cells differentiation. In the present work it was shown that this neuropeptide represents a new natural substrate for Dipeptidyl Peptidase IV (DPPIV). The time-dependent increase of primary amines quantity in the assay mixture of PRP-1 and DPPIV has been observed allowing to conclude, that DPPIV catalyses the enzymatic reaction of PRP-1 cleavage. The amount of primary amines in the assay mixtures was evaluated using o-phtaldialdehyde dye. The gel-filtration and paper electrophoresis analyses proved this conclusion. The catalytic parameters of catalyzed by DPPIV enzymatic reaction of PRP-1 cleavage were determined as: V(max) = 1.27 ± 0.11 nmol/min and K(m) = 0.38 ± 0.1 mM.