Hexokinase isozyme distribution in human skeletal muscle

Two isoforms of hexokinase (type I and type II) are expressed in skeletal muscle; however, the intracellular distribution of these hexokinase isoforms in human skeletal muscle is unclear. The current study was undertaken to assess this issue because binding of hexokinase to subcellular structures is considered to be an important mechanism in the regulation of glucose phosphorylation. Vastus lateralis muscle was obtained from healthy lean individuals. Muscle homogenate was separated at 45,000g into particulate and cytosolic fractions. The activity and subcellular distribution of hexokinase isozymes in human skeletal muscle was determined using ion-exchange chromatography and a highly sensitive high-performance liquid chromatography–based hexokinase assay. This criterion method was used to validate a modified thermal inactivation method for distinguishing type I and type II isoforms. Mean hexokinase activity was 3.88 ± 0.65 U/g wet wt or 0.64 ± 0.11 U/mU creatine kinase (CrK) in the particulate fraction and 0.45 ± 0.22 U/g wet wt or 0.07 ± 0.03 U/mU CrK in the cytosolic fraction. Hexokinase I and II accounted for 70–75 and 25–30% of total hexokinase activity, respectively. Nearly all (95%) of hexokinase I activity (0.52 ± 0.09 U/mU CrK) was found in the particulate fraction, consistent with the known high affinity of hexokinase I for mitochondria. Hexokinase II activity was also largely bound to the particulate fraction (72%), but 28% was found within the cytosolic fraction. Thus, within the particulate fraction, the relative contributions of hexokinase I and hexokinase II were 81 and 19%, whereas within the cytosolic fraction, the relative contributions for hexokinase I and hexokinase II were 37 and 63%.