A TRANSMEMBRANE NADH DEHYDROGENASE IN PORCINE ERYTHROCYTE PLASMA MEMBRANE

NADH dehydrogenase activity has been found in porcine erythrocyte membranes, which appears to be similar to the NADH dehydrogenase activity found in plasma membranes from other cells, such as human erythrocytes, or rat hepatocytes and adipocytes. The specificity, kinetics and other properties of the membrane-bound enzyme have been investigated. The orientation of the dehydrogenase in the membrane has been studied. Determination has been made of redox components present in the membrane, which could function in the dehydrogenase. Detergent has been employed to extract dehydrogenases from the membrane and an enzyme with NADH:ferricyanide reductase and NADH:cytochrome c reductase activities has been partially purified from the extract. The porcine erythrocyte plasma membrane contains an intrinsic dehydrogenase, which displays a specificity for NADH as the electron donor. Various electron acceptors were tested, but the plasma membrane preparation would only reduce DCIP, ferricyanide, cytochrome c and oxygen in the presence of vanadate. NADH:cytochrome c reductase activity has been presumed to proceed through a microsomal type NADH:cytochrome b(,5) reductase. Porcine erythrocyte plasma membrane contains cytochrome b(,5) and P-420, but neither of these cytochromes were reduced by NADH. Even though cytochrome b(,5) was not reduced by NADH, there was still NADH:cytochrome c reductase activity present, indicating that not all the cytochrome c reductase activity can be accounted for by a microsomal type NADH:cytochrome b(,5) reductase. Besides cytochromes b(,5) and P-420 the erythrocyte plasma membrane contains 0.42 nmoles Cu/mg protein and 7.6 nmoles Fe/mg protein. Bathophenanthroline sulfonate, an iron chelator, inhibited the NADH:ferricyanide reductase activity 30%. This may indicate that the iron component in the membrane participates in the NADH:ferricyanide reductase activity. Various other inhibitors were tested and they indicated that the reduction of ferricyanide does not occur at the same site as the reduction of cytochrome c. Right-side-out ghosts and inside-out vesicles can be made from porcine erythrocyte plasma membranes. Oriented vesicle studies indicate that both the cytoplasmic surface and the external surface must be available to the medium in order for the membranes to reveal their total NADH:ferricyanide reductase activity. This indicates that there may be a transmembranous component in the membrane. Treatment of intact erythrocytes with the proteolytic enzyme pronase or with (rho)-diazonium benzene sulfonate (DABS) produced a 20% decrease in NADH:ferricyanide reductase activity in plasma membranes prepared from the treated cells. Intact porcine erythrocytes were able to reduce ferricyanide, and the reduction was shown not to be caused by transport of reducing substances like ascorbate across the membrane. All these facts are evidence that the porcine erythrocyte contains a transmembranous NADH dehydrogenase. A human erythrocyte plasma membrane NADH dehydrogenase has been purified. We partially purified a NADH dehydrogenase from porcine erythrocyte plasma membranes. This partially purified enzyme still showed a specificity for NADH as the reducing agent. The partially purified enzyme was able to reduce ferricyanide, O(,2) (vanadate stimulated) and surprisingly cytochrome c. The fact that cytochrome c can be reduced by the enzyme after partial purification indicates that it is not identical to the microsomal NADH:cytochrome b(,5) reductase. Evidence is presented that indicates that the porcine enzyme is similar to the one isolated from human erythrocytes. It is not clear, as yet, if the partially isolated enzyme is identical to the transmembranous one described here.(,