Hexokinase catalyzes the first step of several metabolic pathways by converting D-hexose to D-hexose-6-P. Hexokinase is an allosteric enzyme inhibited by its products glucose-6-phosphate. At least 4 related hexokinase isoforms (HXKI-III; HXK-IV also known as Glucokinase) have been cloned and characterized. Hexokinases (~100kDa for HXK1-III; HXKIV lacks the N-terminal domains and is ~50 kDa) are outer mitochondrial membrane proteins. The N-terminus, containing the mitochondrial target sequence, and the C-terminal has high sequence homology among various isoforms. The catalytic activity is associated with the C-terminus and other regulatory functions are controlled by the N-terminus.

Human HXKI is a 917 aa (mouse/rat 918 aa) protein. HXK1 is located on chromosome 10. It exists either as a cytosolic protein or as a protein associated with the outer mitochondrial membrane via an interaction with porin, a voltage-dependent anion channel. The enzyme has thus a direct access to the source of ATP generated within the mitochondria. The association of HXK1 with porin is mediated through a highly conserved porin-binding domain (PBD) in the amino terminus of the enzyme. However, recent findings suggest that somatic hexokinases may have additional locations and different functions within cells. For example, hexokinase translocate to the plasma membrane and/or cell cortex of rat macrophages activated by phorbol 12-myristate 13-acetate. Hexokinases may also possess protein kinase activities. Rat brain HXK1 displays protein kinase activity toward exogenous histone 2A in the absence of glucose and also can autophosphorylate on serine, threonine, or tyrosine residues. Defects in HXK1 gene causes hemolytic anemia.

Two cell types, reticulocytes and male germ cells, contain mRNAs encoding variants of HXK1 that do not possess a mitochondrial membrane-binding domain. In human reticulocytes, one of these HXK variants contains an alternative amino terminus that replaces the first 21 residues with 20 alternative amino acids. Interestingly, this reticulocyte-specific HK isozyme does not target to mitochondria but rather is found exclusively in the cytosol. This isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum. In contrast to mouse sperm, human sperm-HXK1 is not tyrosine phosphorylated.

Human, mouse, rat HXKII is a 917 aa protein. It is located on chromosome 2 in humans. HXKII is the predominant isozyme that is expressed in young red cell, the skeletal muscle, adipose tissue, and reticulocytes. It is regulated by insulin. Defects in HXKII are a cause of monogenic autosomal dominant non-insulin-dependent diabetes mellitus type II (MODY-II or NIDDM).

HXKIII (923 aa; chromosome 5; rat 924 aa) has a more limited distribution that includes white cells and shows a genetically determined polymorphism. It is also expressed in lung and liver. It is 50% identical to HXK1.

ADI has produced highly specific rabbit antibodies for various HXKs using antigenic peptide sequences unique to each protein. Respective antigenic or control peptides are also available to confirm specificity of antibodies.