ABSTRACT: Hexokinase type I (HK I; ATP: D-hexose 6- phosphotransferase, EC 2.7.1.1), the predominant glucose- phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ion-exchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.

Keywords: Erythroid hexokinase, red blood cell enzyme, kinetic properties, (human).

Reprint requests to: Professor Mauro Magnani, Istituto di Chimica Biologica "G. Fornaini", Università degli Studi di Urbino, Via Saffi, 2, 61029-Urbino, Italy, phone: 39-722-305211, fax: 39-722-320188, e-mail: magnani@bib.uniurb.it.

ABSTRACT: Human glandular kallikrein (hK2) is a possible new marker for prostate cancer that is homologous to prostate specific antigen. Purified hK2 added to serum or plasma reacted with endogenous protease inhibitors to form complexes of >350, 135, and 80 kDa, and some hK2 remained free, as judged by immunoblotting. The former two complexes could be removed by specific antibodies to alpha2-macroglobulin and to C1- inactivator, respectively, and they comigrated on SDS-PAGE with complexes formed between hK2 and purified alpha2-macroglobulin or C1-inactivator. hK2 complexes of 80 kDa could not be completely removed with any anti-serpin antibody used. Thus, these may consist of more than one type of hK2 complex. In contrast, essentially all hK2 complexes were removed from seminal plasma by antibody to protein C inhibitor, demonstrating that protein C inhibitor is the only significant inhibitor of hK2 in semen. hK2 reacted more rapidly with alpha2-macroglobulin than with any other inhibitor in plasma or serum. Divalent metal ions and heparin did not appreciably affect the rate of formation of any of the hK2 complexes in serum or plasma or with purified alpha2- macroglobulin or C1-inactivator. Measurement of one or more of the hK2 forms identified here may have diagnostic or prognostic potential for prostate cancer.

Keywords: Prostate cancer, human glandular kallikrein, alpha2-macroglobulin, C1- inactivator, protein C inhibitor, benign prostatic hyperplasia.

Reprint requests to: Mary J. Heeb, Ph.D., Department of Molecular and Experimental Medicine (SBR5), The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, phone: (619) 784-2185, fax: (619) 784-2243, e-mail: heeb@scripps.edu.