ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 347, No. 1, November 1, pp. 69–77, 1997 Article No. BB970310 Rat Harderian Gland Porphobilinogen Deaminase: Characterization Studies and Regulatory Action of Protoporphyrin IX Carina A. Cardalda, Adela Ana Juknat, Fernando G. Princ, and Alcira Batlle 1 Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP) (CONICET), Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires Ciudad Universitaria, 1428 Buenos Aires, Argentina Received May 6, 1997, and in revised form July 25, 1997 ian gland; cysteine residue(s); protoporphyrin IX; por- phobilinogen deaminase regulation. Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer of M r 38 { 2 kDa and is optimally active at pH 8.0–8.2. Its The harderian glands, present in most terrestrial activation energy, determined by an Arrhenius plot, is vertebrates, are large paired lipid-secreting intraor- 76.1 kJ/mol. Initial velocity studies showed a linear bital structures (1, 2). The main function of the hard- progress curve for uroporphyringen I formation and a erian gland is yet unknown and it is unclear whether hyperbolic dependence of the initial rate on substrate the gland can act both as an exocrine and as an endo- concentration, indicating the existence of a sequential crine organ. However, it has been proposed that the displacement mechanism. Apparent kinetic constants, harderian gland is involved in many diverse func- K m and V m , calculated at 37°C and pH 8.0 were 1.1 mM tions such as lubrication of the eyes, photoprotection, and 170 pmol/min mg, respectively. The pH depen- production of pheromones, and thermo- and osmoreg- dence of the apparent kinetic parameters revealed the ulation (see Ref. 3 for review). Moreover, the harder- ionization of residues with pK ES A and pK ES B of 7.4 { 0.1 ian gland is considered to be a site of immune re- and 8.6 { 0.1, respectively, and a pK E value of 8.0 { 0.1. sponse, a source of growth factors and a part of the Incubation of PBG-D with 5.0 mM N-ethylmaleimide retinal – pineal axis (see 3 for review). A very interest- and 5.0 mM 5,5-dithiobis(2-nitrobenzoic acid) at pH 8.0 ing characteristic of these glands is that in many led to inhibitions of 70 and 50%, respectively. The ef- rodent species, they synthesize and store large quan- fect of pH, as well as the effect of thiol reagents, on tities of porphyrins, located at intraluminal, intersti- enzyme activity strongly suggests the involvement of tial, and intracellular levels (4). In rodents, the main cysteine residue(s) in the mechanism of uroporphyri- porphyrin present is the heme precursor protopor- nogen I biosynthesis, in both the catalytic reaction and phyrin IX with smaller amounts of coproporphyrin the substrate binding. Rat harderian gland PBG-D ac- and the tricarboxylic so-called harderoporphyrin (1, tivity decreased with increasing concentrations of protoporphyrin IX, reaching a 40% inhibition at the in 5–7). Although the harderian glands have been ex- vivo concentration of the porphyrin and 7 mM PBG. tensively studied in the last years (3, 8), the biological Even at saturating concentrations of substrate, inhibi- role of accumulated porphyrins is not known and the tion by protoporphyrin was not completely reversed. regulatory mechanisms of porphyrin metabolism are So, accumulated porphyrin may act as an regulator of yet to be determined. PBG-D activity in rat harderian gland.  1997 Academic Moore et al. (9) reported that in female hamster hard- Press erian gland, the activity of d-aminolevulinate synthase Key Words: porphobilinogen deaminase; rat harder- (ALA-S; EC 2.3.1.37), 2 the rate-limiting enzyme in por- 2 Abbreviations used: ALA, d-aminolevulinic acid; PBG, porphobili- 1 To whom correspondence should be addressed at Viamonte 1881, nogen; ALA-S, d-aminolevulinic acid synthetase; ALA-D, d-aminolev- ulinic acid dehydratase; PBG-D, porphobilinogen deaminase; HMB, 10 Piso, Dto. A, 1056 Buenos Aires, Argentina. Fax: 54 1 811 7447. E-mail: cipyp@alad.fcen.uba.ar. hydroxymethylbilane; URO-D, uroporphyrinogen decarboxylase; 69 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.