Human erythrocyte hexokinase deficiency. Characterization of a mutant enzyme with abnormal regulatory properties.

Abstract

A B S T R A C T In the erythrocytes of a patient with hereditary nonspherocytic hemolytic anemia, a homozygous expression of hexokinase deficiency was detected. The mutant enzyme was characterized by normal kinetic parameters with respect to its substrates, glucose and MgATP2-, normal pH optimum, normal heat stability at 40°C, but abnormal behavior with respect to its regulation by glucose-1,6-diphosphate and inorganic phosphate, and an altered electrophoretic pattern. Interpretation of the results revealed the presence of two different hexokinases type I in normal human erythrocytes: one enzyme with a high affinity for glucose-1,6-diphosphate, the inhibition of which is regulated by inorganic phosphate; and another enzyme with a lower affinity for the inhibitor, not regulated by inorganic phosphate. The former enzyme was not detectable in the erythrocytes of the patient, whereas the presence of the latter enzyme could be demonstrated.

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

“…The presence of at least two subtypes of hexokinase type I with different regulatory properties was suggested for erythrocyte hexokinase [17] and confirmed by Vowles and Easterby [24] for hexokinase I from pig heart. Their results show that the activity of one form is overcome by Pi after glucose-6-phosphate inhibition more than the other.…”

“…Their results show that the activity of one form is overcome by Pi after glucose-6-phosphate inhibition more than the other. It was hypothesized by Rijksen and Staal [17] that a phosphate-insensitive form of hexokinase I (called type Ic) is formed out of a phosphate sensitive form (form Ib) by some post-synthetic modification. According to this hypothesis, it might be possible that the modification process in the leukemic CEM cells works at an elevated level in comparison to normal lymphocytes and non-leukemic lymphoblasts.…”

“…Blanks stains in the absence of either glucose or ATP were carried out for control staining. Polyacrylamide gel electrophoresis using a discontinuous buffer system was performed as described previously [17]. Staining was carried out in the same mixture as used for the staining of the cellulose acetate strips with omission of the agar.…”

“…The poor separation between the bands does not allow exact identification of the particular isozymes. CEM cell hexokinase I consists mainly of two subisozymes which are presumably type Ia and Ic, according to the nomenclature of Rijksen and Staal [17]. In normal lymphocytes as well as in stimulated lymphoblasts two main bands and a minor one were present.…”

“…Inorganic phosphate (Pi) is capable of overcoming this inhibition in part [15--17]. Three subisozymes of hexokinase I, designated Ia, Ib and Ic [17], may exist. It is hypothesized, that Ic is formed from Ib by a posttranslational modification.…”