NADH-diaphorase Activity Research Articles

Distribution studies on polyglutamate forms of folate.

this case was found to be 42.0s' (see above). The effects on other NADH diaphorase activities of arsenite treatment of reduced enzyme are shown in Table 1. In direct contrast with its effects on oxidized enzyme (Fig. l), is the marked inhibition of NADHdichlorophenol-indophenol activity accompanying arsenite treatment of NADHreduced enzyme. This could be due to the exposure on reduction of a second site with which arsenite may interact (Rajagopalan &Handler, 1967). However, in agreement with the data of Fig. 1 (b), arsenite treatment of reduced-cyanide-inactivated enzyme failed to alter activity significantly (Table 1). Thus we suggest that pre-reduction merely alters the mode of binding of arsenite to the active-centre persulphide groups. The effects of arsenite on the NADH diaphorase activity of oxidized or reduced enzyme clearly depend on the integrity of the molybdenum-centre persulphide groups and are restricted to activity with dichlorophenol-indophenol and trinitrobenzene sulphonate as electron acceptors. This, in our view, infers that these two acceptors also interact with the molybdenum centres of these enzymes.

Diaphorase activity located in inclusions induced by soil-borne wheat mosaic virus.

Extracts of rye leaves infected with soil-borne wheat mosaic virus, isolate US-B, were examined with the electron microscope. They contained inclusions consisting of membrane bound tubules. Partially purified inclusions had the same fine structures as that observed in thin sections of leaf cells. Inclusions in epidermal strips and partially purified preparations were examined cytochemically. They had an intense diaphorase activity (NADH or NADPH nitrobluetetrazolium oxidoreductase). The activity was lost after heating at 55° for 5 min, fixation with ethanol or glutaraldehyde, and incubation with 5 × 10−5 M parachloromercuribenzoate. The activity was intense when NADH was used as a hydrogen donor, but was weak with NADPH. The cytoplasm of leaf cells also had similar diaphorase activity although the activity was weak. When diaphorase activity in various cellular fractions was assayed, about 60–70% of the activity was in the soluble fraction, 13–14% was in the 15,000 g pellet, and 1–3% in the microsomal fraction. The ratio of specific activity of NADH diaphorase to NADPH diaphorase was about 2 in the microsomal fractions but was 0.6–0.9 in the other fractions. Microsomal fractions from infected leaves contained fragments of inclusions and had higher ratios of NADH to NADPH diaphorase activity than those from uninfected leaves. Partially purified inclusions had higher NADH diaphorase activity than NADPH diaphorase. These results indicated that diaphorase activity was bounded to inclusion tubules and that the activity had properties in common with endoplasmic reticulum.

Postnatal development of acetylcholinesterase in the caudate-putamen nucleus and substantia nigra of rats

The postnatal development of acetylcholinesterase (AChE, EC 3.1.1.7) and NADH-diaphorase was examined in the caudate-putamen nucleus and substantia nigra of rats ranging from 3 to 90 days in age. From 3 to 15 days post partum islands of AChE and NADH-diaphorase activity were observed in the caudate-putamen nucleus. Individual neuronal somata could also be seen in AChE-stained sections up to 15 days. At later ages neuropil staining became increasingly dense, and this presumably accounted for the infrequent visualization of cell bodies in the brains of older animals. During development AChE appeared in the caudate-putamen nucleus in a lateral to medial topographic order; analogously, enzyme staining in the neostriatum reappeared in the same lateral to medial topographic order in adult rats following irreversible AChE inhibition by intramuscularly injected bis-(1-methylethyl)phosphorofluoridate (di-isopropylfluorophosphate: DFP). Furthermore, DFP treatment in mature animals revealed the presence of AChE in striatal neurons having morphologies similar to those observed in newborn rats. A similar time-course of postnatal AChE development was observed in the substantia nigra. In both the pars compacta and pars reticulata individual cell bodies, which were visible at early ages (3-10 days), became increasingly obscured at later times after birth by extra-somata staining. Between the 6th and 15th postnatal days AChE-containing fibers were seen projecting apparently from pars compacta into pars reticulata. Comparison of the present results with histochemical data of other investigators on the postnatal development of monoamines indicated the likelihood of cholinergicmonoaminergic interactions in the neostriatum and substantia nigra.

Nitrate reductase from Spinacea oleracea. FAD and the inactivation by NAD(P)H

Treatment by p-hydroxymercuribenzoate of nitrate reductase from spinach leaves causes the disappearance of NADH-diaphorase activity and the appearance of an FAD-requirement for the inactivation by NAD(P)H of FNH 2-nitrate reductase. The diaphorase activity of the treated preparation is not affected by incubation with FAD or the addition of this nucleotide to the assay mixture. Conversely, filtration of the native preparation through a column of Sepharose 6B produces the appearance of an FAD-stimulation of the diaphorase activity, but no effect of FAD on the NADH-inactivation was observed. These differences between the FAD-requirement of NADH-diaphorase activity and NADH-inactivation agree with the postulated independence of the two processes.

A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa.

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.

Fast fixation of brain in situ by high intensity microwave irradiation: application to neurochemical studies.

The levels of acetylcholine and choline were measured in various brain regions of the rat after fixation by microwave irradiation of the head and after decapitation and subsequent freezing in liquid nitrogen. Levels of acetylcholine were increased by approximately 50% after microwave irradiation, while choline levels were reduced. These biochemical findings were correlated with virtually complex loss of acetylcholinesterase and NADH-diaphorase activity after 1 s exposure to microwave irradiation at a level of 5 kW.

Extraocular muscles light microscopy and ultrastructural features

Thirty extraocular muscles (EOM) from 20 patients were evaluated by light microscopy (LM), electron microscopy (EM), and enzyme histochemistry (EZH). Twenty-one EOM were obtained from 13 patients with strabismus, 9 EOM from 4 patients undergoing eye surgery for other reasons and from 3 autopsy cases. One mum thick sections revealed marked variation in muscle fibre shape and size and in myofibrillar structure; also noted were small, hypertrophied, whorled, and ringbinden fibres. Dense and granular material in the central portion of some fibres and sarcomere disruption in 2--3 mum sections was observed. EZH revealed the absence of the classical mosaic pattern usually found in skeletal muscles. ATPase studies were inconsistent and did not correlate with the expected reciprocal activity of NAD-H diaphorase, particularly on the large fibres. Ultrastructural features consisted of vacuoles within myofilament bundles, "smearing" of Z bands, and "nemaline rods". Occasional myelin figures and lipid-like droplets were observed in subsarcolemmal spaces, associated with scattered clusters of glycogen granules. Abnormal mitochondria and subsarcolemmal inclusions of dense and granular material were conspicuous. "Leptomeric" profiles, "Zebra bodies", or "striated bodies" were noted in 8 EOM's, and an Hirano body was found in 1. The intramuscular nerves contained structures resembling "Luse bodies" in 7 cases. These observations suggest that EOM from individuals with and without strabismus possess unique structural characteristics suggestive of developmental and morphological disarrangement of contractile elements. Some of these changes might play a role in the pathogenesis of strabismus and in the development of clinical symptoms. These features are significantly different from striated skeletal muscle. Therefore the criteria used in the pathological evaluation and diagnosis of skeletal muscle disorders cannot be unequivocally applied to EOM investigations. These data establish the necessity to determine histological norms, ultrastructural patterns, and develop new enzyme histochemistry criteria for the evaluation of EOM. Only then can an acceptable comparison of EOM and skeletal muscle be made.

Die Verwendbarkeit einer Niederdruckperfusionsmethode für enzymhistochemische untersuchungen der Cochlea, dargestellt am Beispiel der NADH-Diaphorase

The use of a low pressure perfusion method for enzyme histochemical reactions in the cochlea of guinea pig is proved in the case of the demonstration of the NADH-diaphorase. It was shown by measuring of the microphonic potentials during the perfusion, that the perfusion medias caused practically no chemical or mechanical defections of the hair cells under the new conditions of working. The activities of the NADH-diaphorase were demonstrated mainly in the hair cells and in the nervous fibres of the organ of Corti. A remarkable NADH-diaphorase activity was detected in the Boettcher cells too.

Nitrate reductase from Spinacea oleracea. Effects of sulfhydryl-group reagents on the activities of the complex and the inactivation by NADH

Sulfhydryl-group reagents inactive the nitrate reductase complex from Spinacea oleracea. Most of the reagents used inactivate selectively the NADH-diaphorase moiety. However, at higher concentrations of reagent the FNH 2-nitrate reductase is also affected. Enzyme preparations inactivated by p-hydroxy-mercuribenzoate can be reactivated by dithioerythritol. Nitrate reductase lacking NADH-diaphorase activity, after treatment with p-hydroxymercuribenzoate, is inactivated in its FNH 2-nitrate reductase moiety by NADH in the same way as the untreated preparation. This apparent independence of the NADH-inactivation process from NADH-diaphorase activity supports the postulated existence of a binding site for pyridine nucleotides implicated in NADH-inactivation and different from the diaphorase catalytic site.

Histochemical profiles of rat soleus intrafusal fibres after chronic exercise.

Intrafusal fibres from the rat soleus were investigated for representative histochemical profiles in sedentary animals and animals chronically exercised for 17 weeks on a treadmill. The pattern of myosin adenosine triphosphatase (ATPase) activity in the polar region revealed three intrafusal fibre types: (1) myosin ATPase-dark (MD) fibres, alkali- and acid-stabile; (2) myosin ATPase-light (ML) fibres, alkali- and acid-labile; and (3) myosin ATPase-reversible (MR) fibres, alkali-stabile and acid-labile. The three fibre types were correlated with the level of reduced NADH diaphorase activity, with MR, ML and MD fibres staining dark, moderate and light, respectively. In the equatorial region the morphological features of representative ML and MD fibres revealed that they were nuclear bag fibres, while representative MR fibres were identified as nuclear chain fibres. The MR fibres in the exercised animals had higher levels of myosin ATPase alkaline stability and acid lability than MR fibres in the sedentary animals, suggesting the MR fibre profiles are selectively influenced by chronic exercise. The mean cross-sectional area of MR fibres from the exercised animals was significantly less than the MR fibres from the sedentary animals. In contrast to the effect of endurance training on NADH diaphorase activity in extrafusal muscle fibres, there was evidence of less activity in the MD fibres of the exercised animals.

Focal loss of alkaline phosphatase and increase of proliferation in preneoplastic areas of the rat urothelium after administration of n-butyl-n-(4-hydroxybutyl)-nitrosamine and n-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide.

Development of tumours of the urinarY bladder was studied in 59 Male and female Sprague-Dawley and Wistar rats with combined enzyme-histochemical and autoradiographic methods after oral application of n-butyl-n-(4-hydroxybutyl)-nitrosamine (BBN) and n-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT). as the first carcinogenic lesion detectable by light-microscopy a focal, sharply defined irreversible loss of alkaline phosphatase activity was consistently demonstrated in the urothelium, which appeared normal histologically and cytologically. In about 2/3 of the cases, NADH-diaphorase activity was markedly reduced in identical regions. The enzyme-deficient areas are to be considered as preneoplastic, because papillomas and carcinomas developed from them through different stages of hyperplasia. As a rule, these also were characterized by total loss of alkaline phosphatase activity and attenuation of the NADH-diaphorase in all parts or circumscribed areas. Autoradiographically 3H-thymidine-labelling index revealed a 43.2-fold (BBN) and 22.6-fold (FANFT) increase, respectively, in the enzyme-deficient areas, as compared with the surrounding emzyme-containing urothelium. After 54 hrs of continous labelling, there was a mean 3H-thymidine-labelling index of 54.9% in the enzyme-negative regions. The physiological mode of regeneration was no longer maintained in the areas of enzyme deficiency as there was an increased proliferation of suprabasal cells. Areas of papillomas that showed a marked attention of NADH-diaphorase had a 3H-thymidine-labelling index 4.5 (BBN) and 3.1 (FANFT) greater than the surrounding areas with preserved enzyme activity. Since loss of alkaline phosphatase activity occurs regulary and consistently after application of carcinogens with chemically different structures it appears to indicate the initial phase of tumor development in the urinary bladder of the rat.

Functional Morphology of Steroidogenic Tissue and Adrenomedullary Cells in the Adrenal Gland ofRana tigrina(Daud.) andRana cyanophlyctis(Schn.)

The presence of δ5-3β-HSDH, 17β-HSDH, 11β-HSDH, G-6-PDH and DPNH-diaphorase activity has been demonstrated in the interrenal cells of two frogs, R. tigrina and R. cyanophlyctis. The substrate specificity of δ5-3β-HSDH and 17β-HSDH was tested by utilizing different specific hydroxysteroids. DL5-3β-HSDH, G-6-PDH and NADH-diaphorase activity was relatively more in the peripheral region of the interrenal tissue compared to the cells in the middle region, apparently indicating a zonation of the adrenocortical tissue. The presence of 11β-HSDH, G-6-PDH and NADH-diaphorase has been also observed in the renal tubules which indicates that the renal tubules might convert hydroxysteroids to ketosteroids during steroid excretion. The presence of two types of adrenal medullary cells showing positive iodate and chromate reactions was observed.

Reversal of age-related changes in microsomal enzyme activities following the administration of triamcinolone, triiodothyronine and phenobarbital

1. 1. Microsomes were isolated from the livers of 3- and 24-month-old Fischer rats, and specific activities were determined for glucose-6-phosphatase, NADPH cytochrome c reductase, NADPH diaphorase, NADH cytochrome c reductase, NADH diaphorase and NADH neotetrazolium reductase. 2. 2. In agreement with other reports the specific activities of glucose-6-phosphatase and NADPH cytochrome c reductase decreased in the old animals, and NADH cytochrome c reductase increased. 3. 3. Chronic administration of low doses of triamcinolone almost doubled the specific activity of glucose-6-phosphatase in old animals, but caused essentially no change in young animals. In contrast, treatment with phenobarbital halved this activity in young animals, but was essentially without effect in old animals. Treatment with triiodothyronine and caused a marked stimulation of this activity in both young and old animals. 4. 4. There was very little change in the specific activity of NADPH cytochrome c reductase or NADPH diaphorase following these treatments. 5. 5. NADH cytochrome c reductase, NADH neotetrazolium reductase and NADH diaphorase activities were greatly decreased in both young and old animals by administration of all three compounds. 6. 6. Age-related differences in the activity of these enzymes were either diminished or reversed following each treatment. Old animals were as responsive to these stimuli as young animals, and the highest and lowest specific activities observed for each enzyme during the course of all experiments were very similar for young and old animals of the same sex. These observations suggest that age-related differences in these enzyme activities are probably caused by a different metabolic or endocrine state in young and old animals or their hepatocytes, and not by a deterioration of the integrity or control of the synthesis of these proteins.

Pitfalls in histochemical localization studies of NADPH generating enzymes or enzyme systems in rat small intestine.

In the presence of excess β-glycerophosphate or p-nitrophenylphosphate NADPH diaphorase of the epithelial cell in rat small intestine has its highest activity in the villi at the basis of the microvilli, where smooth endoplasmic reticulum of the cells is present. In the absence of β-glycerophosphate or p-nitrophenylphosphate the diaphorase activity is much higher and broader localized in crypts and villi. The increased activity is due to the conversion of NADPH to NADH by non-specific (mostly alkaline-) phosphatase activity, so that both NADPH and NADH diaphorase activities are measured. When NADPH is generated by a specific NADP+-linked dehydrogenase, such as glucose-6-phosphate dehydrogenase, and nitroblue tetrazolium is present to trap the reducing equivalents formed, the histochemical localization of the dehydrogenase is identical to that of NADPH diaphorase, although the dehydrogenase may be present in another cell compartment (glucose-6-phosphate dehydrogenase for instance has a cytosolic distribution). Therefore the localization of a dehydrogenase may be falsely interpreted histochemically, according to the diaphorase reaction involved. A different localization may be obtained when the diaphorase reaction is circumvented by the addition of an alternative hydrogen carrier, such as phenazine methosulphate. Also in coupled dehydrogenase assays this may be observed. The hexokinase reaction, coupled to the glucose-6-phosphate dehydrogenase reaction in the presence of nitroblue tetrazolium and the absence of phenazine methosulphate, has a distribution identical to NADPH diaphorase. In the presence of phenazine methosulphate the enzyme has an almost ubiquitous distribution in the small intestinal epithelial cell. When a substrate may react both with NADP+- and NAD+-linked dehydrogenases, such as L-malate: NADP oxido-reductase (decarboxylating) and L-malate: NAD oxidoreductase respectively, the high activity of intestinal alkaline phosphatase may influence the histochemical distribution by converting part of NADP+ to NAD+. The addition of another phosphate ester, such a β-glycerophosphate or p-nitrophenylphosphate, may therefore influence the observed distribution.

Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm.

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b(5) and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm(3) and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm(3) and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.

Effect of hind-limb immobilization on contractile and histochemical properties of skeletal muscle.

Both hind limbs of male rats were immobilized in casts. After 4 weeks, serial sections of hind limb muscles were stained for myosin ATPase and NADH-diaphorase. The soleus from immobilized limbs had significantly fewer muscle fibers than the control soleus. Moreover, the soleus from immobilized limbs had a significantly lower percentage and lower number of fibers with low myosin ATPase activity than the soleus from control rats. Immobilization also resulted in the speed of contraction for the soleus being significantly faster than the soleus from control rats. There were no significant differences in the contractile properties or in the percentages of fibers with low myosin ATPase between rectus femoris muscles from immobilized and control limbs. The deep portion of the rectus femoris from immobilized limbs had a significantly smaller percentage of muscle fibers with high NADH-diaphorase activity than did the rectus femoris from control rats.

Congenital Methemoglobinemia: The Result of Age-dependent Decay of Methemoglobin Reductase

Abstract Methemoglobin reductase activity (MHR), measured by the Hegesh assay, decreases as normal red cells age. This probably accounts for the slighlty increased concentration of methemoglobin in older red cells. Exaggeration of normal MHR age lability was demonstrated in several individuals with various molecular abnormalities of NADH-diaphorase and was associated with more striking accumulation of methemoglobin in the older red cells of those patients. Diaphorase activity, measured by the Scott assay, does not exhibit age lability in normal red cells or the cells of affected patients. While diaphorase uses either NADH or NADPH as a cofactor, methemoglobin reductase requires NADH. Methemoglobin reductase is found in hemolysates but not in membrane preparations, while approximately 1% of total red cell NADH-diaphorase activity is membrane-associated. The specific activity of NADH-diaphorase is approximately 60 times greater than that of methemoglobin reductase. Whether the two activities represent distinct enzyme proteins or differing substrate affinities of the same enzyme awaits adaptation of the Hegesh assay to enzyme electrophoresis.

Studies on Methemoglobin Reductase: I. COMPARATIVE STUDIES OF DIAPHORASES FROM NORMAL AND METHEMOGLOBINEMIC ERYTHROCYTES

Abstract Nonhemoglobin proteins of normal human red cells and methemoglobinemic red cells were separated from hemoglobin by carboxymethylcellulose column chromatography and NADH-dependent diaphorases were fractionated by the column chromatography on Sephadex G-75 and DEAE-Sephadex. Two NADH-dependent diaphorases, diaphorase A and diaphorase C, were isolated from the normal red cell extracts, diaphorase A forming 90% of the total activity. The Km of partially purified diaphorase A for 2,6-dichloroindophenol is 3.3 x 10-4 m. The diaphorase A fraction was less than 3% of normal in the methemoglobinemic red cell extracts. The methemoglobinemic enzyme is similar to the normal enzyme with respect to its Km for the dye, affinity with NADH, and the effects of phosphate on its activity, and, although its chromatographic behavior is normal, it is not identical with the normal enzyme, judging from its heat stability and electrophoretic properties. When assayed in 1.0 m sodium phosphate buffer, another NADH-dependent diaphorase, diaphorase B, is seen in both the normal and methemoglobinemic red cell extracts, which is found to be essentially NADPH-dependent diaphorase. It exhibits NADH-diaphorase activity in proportion to phosphate concentration and causes the phosphate activation of NADH-diaphorase activity in the methemoglobinemic hemolysate. When assayed in 1.0 m phosphate buffer, Km for the dye is 2.0 x 10-5 m and Km for NADH is 9.0 x 10-5 m regardless of NADH and the dye concentrations, respectively, which suggests a reaction mechanism whereby the affinity of one substrate to the enzyme is not influenced by the presence of another substrate.

Chemical and enzymatic characteristics of cytoplasmic membranes of poliovirus-infected HeLa cells

The density distribution of cytoplasmic membranes separated by centrifugation to equilibrium in discontinuous sucrose gradients is progressively altered after infection of HeLa cells with poliovirus. The most striking changes are a decrease in quantity and an increase in density of rough microsomes and a large increase in quantity of the smooth microsomal membranes with which viral RNA synthesis is associated. The smooth membranes begin to increase between 2.5 and 3.5 hr after infection, and continue to proliferate late into infection. They differ from the membranes in the corresponding fraction of uninfected cells in their higher phospholipid:protein ratio, altered phospholipid composition, and reduced specific activity of NADH diaphorase. The specific activities of several enzymes were determined in membrane fractions obtained from discontinuous sucrose gradients. Mitochondrial and lysosomal enzyme activities are found mainly in a fraction which also contains most of the rough endoplasmic reticulum. However, 4 hr after infection a lysosomal enzyme, β-glucuronidase, is distributed throughout the gradient. Most of the plasma membrane fragments are sedimented in the low speed centrifugation prior to layering into the discontinuous sucrose gradients. The small proportion of the plasma membrane fragments that are layered into the gradients cosediment with the smooth microsomal membranes.

Age lability of normal and variant methemoglobin reductase

The concentration of methemoglobin increases as red cells age in normals and in patients with congenital methemoglobinemia (CMHb), yet the activity of NADH diaphorase (ND) (Scott assay) does not decrease with age. A specific assay for methemoglobin reductase (MHR) (J. Lab. Clin. Med. 72.339, 1968) permits reassessment of this phenomenon. Red cells were separated by centrifugation at 100,000 g for 1 hour. The top and bottom 10% were harvested and mean ages determined by glutamic oxaloacetic transaminase (GOT) activity. The age stability of ND activity was confirmed. The MHR activity of old cells (bottom layer) was 15% lower than that of younger (top layer) cells in normal individuals. In one family with CMHb and two fast migrating isoenzyme variant of ND, the activity was only slightly reduced (12%) whereas MHR activity was markedly reduced (40 to 90%) in the old cells compared to young cells. An unrelated female with CMHb and a slow migrating variant of ND was previously reported to have “pseudo-mosaicism” on the basis of heterogeneous distribution of methemoglobin between her younger and older cells (NEJM 275:397, 1966). Her older cells actually had greater activity of ND than did her younger cells. In contrast, the MHR activity of her older cells was only 10% of her younger cells. Thus, the normal age-lability of MHR can account for the accumulation of methemoglobin as erythrocytes mature. Exaggeration of this tendency due to structural modifications in the enzyme molecules may account for methemoglobinemia in patients carrying variant isoenzymes.