An interpretation of human diaphorase isozymes in terms of three gene loci DIA₁, DIA₂ and DIA₃

Abstract

NADH and NADPH diaphorase isozymes have been studied in human tissues. Evidence from rare heterozygotes suggests that the red cell and main tissue forms of NADH diaphorase are products of the same locus DIA1. NADPH-dependent diaphorase appears to be the product of a second locus DIA2. A third locus, DIA3, codes for the polymorphic sperm diaphorase. The products of this locus are also found in foetal tissues including placenta and adult brain and gonads. The products of these three loci may be distinguished by their substrate specificity, thermostability and molecular size.

Human FAD-dependent NAD(P)H diaphorase

“…Diaphorase-4 is clearly distinguished from other human diaphorases by its dependence on FAD for activity after electrophoresis, which presumably reflects a rather weak association with the coenzyme, by its inhibition with dicoumarol, by its high affinity for Cibacron Blue and by its molecular size. The molecular weight of diaphorase-4 is estimated to be close to 50000, whereas diaphorase-1, diaphorase-2 and diaphorase-3 have molecular weights of 30000, 18000 and 30000 respectively (Fisher et al, 1977). Furthermore, somatic cell hybrid studies (Grzeschik, 1979;Povey et al, 1980) suggest a dimeric structure for diaphorase-4 that is in contrast with the other diaphorases, which are monomeric.…”

“…Diaphorase-1 was eluted, as expected, in a volume corresponding to a molecular weight of 30000 (Fisher et al, 1977), and two other diaphorases, designated diaphorase-5 and diaphorase-8, were eluted at positions corresponding to molecular weights close to 135000 and 115000 respectively. These two diaphorases have so far only been detected in kidney, and diaphorase-5 is exceptional since it apparently does not utilize 2,6-dichlorophenol-indophenol and is only detected with menadione as electron acceptor.…”

“…A complex series of enzymes catalysing the oxidation of NAD(P)H and using the quinoid redox dye 2,6-dichlorophenol-indophenol as electron acceptor have been described in human tissues (Leroux & Kaplan, 1972;Fisher et al, 1977). Since most tissues contain a heterogeneous collection of such diaphorases, analysis of crude material by spectrophotometric assay can be misleading and relatively uninformative.…”

“…Each of these diaphorases is detected after electrophoresis as a series of active components. Detailed genetic and biochemical studies have shown that in all three cases the component isoenzymes are determined by the same gene and represent the major gene product and a series of chemically modified forms generated post-translation (Fisher et al, 1977;Edwards et al, 1979.) The physiological substrates of the various diaphorases have not yet been identified with certainty, but in erythrocytes diaphorase-1 is the major cytochrome b5 reductase, with an important role in methaemoglobin reduction (Hultquist & Passon, 1971: Leroux et al, 1975.…”

NADH cytochrome b5 reductase activity in lymphoid cell lines. Expression of the defect in epstein Barr virus transformed lymphoblastoid cell lines from patients with recessive congenital methemoglobinemia.

“…However, in type I RCM, the enzyme activity was significantly lower in LCL than in leukocytes. We have no explanation for this phenomenon, which could be accounted for by either differential stability of the DIA, product, or by differential expression of the diaphorase isozymes (32). In the LCL from type II RCM, the enzyme defect was much more pronounced than in type I cells (Table IV), as already found with other nucleated cells (7,8,27,28).…”