G-6-PDH

The conclusion that the significance of the pentose phosphate shunt may be in keeping NADP in its reduced state and furnishing pentose phosphates for biosynthesis, rather than G-6-P utilization, is closely confirmed by the fact that individuals deficient in or lacking G-6-PDH activity suffer from a number of metabolic disorders due to lack of NADPH2 generation and nucleotide depletion. 

Both G-6-PDH and 6-PGDH have been found in tissues of plants, microorganisms, and animals, suggesting the widespread importance of the oxidative pathway of glucose metabolism. 

G-6-PDH and 6-PGDH were isolated from yeast (G9, H15, H19, K19, Nl, N4), from Neurospora crassa (R3), from Escherichia coli (M12), from Aspergillus flavus-oryzae (H10), and from Aspergillus niger (J2). 

The enzymes have been demonstrated to be present in varying amounts in almost all mammalian tissues and in blood (B4, D4, D7, D8, G8, G10, Gil, H13, S12, S14, W5, W18). The highest activities are attributed to erythrocytes and to the other cellular components of blood. Recently, G-6-PDH and 6-PGDH activities in saliva have been described (R5). 

Generally, both G-6-PDH and 6-PGDH are regarded as being NADP specific (B4, C2, D7, D8, H13, H15, H16, N2, R12, W5, W6, W18). Nevertheless, the interaction of NAD in the reactions of Eq. (3) and (5) has been reported (C2, H10, L5), especially if high concentrations of NAD were used as a hydrogen acceptor (N2). The role that NAD plays in these reactions remains unexplained, but it may be of less importance under steady state conditions. 

In Table 2 is given a synopsis of known inhibiting and activating compounds for G-6-PDH and 6-PGDH. 

The inhibition of G-6-PDH and 6-PGDH by heavy metal ions and its reversibility by adding EDTA demonstrates the requirement of active sulfhydryl groups (GIO, R6). This finding is confirmed by the activating action of cysteine (N2, W18). However, an inhibition of... 

Material G-6-PDH 0-PGDH Remarks on assay method References... 

G-6-PDH activity in the presence of EDTA + Mg+ + ions has been reported (M8), suggesting the probable formation of an inhibitory EDTA-Mg-substrate complex. Nevertheless, addition of EDTA may be important in the estimation of serum activities, since the levels of heavy metals (e.g., in hepatitis) may be elevated to inhibiting amounts, thus resulting in false values. Cu++ and Zn++ ions have been found to inhibit exclusively 6-PGDH activity without affecting G-6-PDH activity (GIO). 

Galactose-l-phosphate has been found to inhibit G-6-PDH but not 6-PGDH (L2). This finding is important in galactosemia in which galactose-l-phosphate accumulates because of lack of p-galactose uridyl transferase in tissues and plasma. 

ATP inhibits only 6-PGDH and not G-6-PDH (G10). As previously reported for other enzymes, phenothiazine derivatives inhibit both G-6-PDH and 6-PGDH competitive with NADP since NADP protects G-6-PDH depending on its concentration (C4). Neither inhibiting nor activating action has been observed in phenylbutazone treatment (F2). 

Reduced G-6-PDH activity entails in intact rats a longer starvation period, thus suggesting an influence of pituitary function on maintenance of normal G-6-PDH level, or on the utilization of G-6-P by the oxidative pathway. 

Fig. 5. G-6-PDH activity in liver cell homogenates of rats under physiological and pathological conditions. Activity in livers of normal fed animals = 100%. Cf. (W10). 

Adrenalectomy causes a decrease of G-6-PDH activity in liver homogenates of rats. This can be restored to normal levels by administration of cortisone or corticosterone while hydrocortisone and progesterone dosages resulted in a partial restoration of activity (W17). 

A correlation between the activity of G-6-PDH and extractable renin in rat kidneys has been reported, suggesting that the macula densa cells and also the juxtaglomerular apparatus are parts of a system related to the formation of renin (H6). 

The mechanisms of all these alterations in G-6-PDH and 6-PGDH activities are as yet unknown and will have to be clarified. A special question is whether or not we can consider them as real hormonal control or as a simple coincidence conditioning alterations in equilibria of enzyme reactions involved. 

Decreasing G-6-PDH activity in aging erythrocytes has been described (L4, L8). This finding becomes important in hereditary G-6-PDH deficiency, explaining the self-limiting effect of drug-induced hemolysis (B9, BIO, Bll). 

Investigations of changes in G-6-PDH activity of liver cells in starving rats resulted in the finding of reduced activity (A3) depending on the duration of the starvation period (W10). 

The G-6-PDH activity of erythrocytes of human newborns was found to be approximately 100% higher than in healthy children and adults, although the red cells were GSH unstable (Bl) (cf. Section 3.4). 

The determination of G-6-PDH and 6-PGDH activities is possible by 3 ways (a) by measuring the 02 uptake when the respective reaction product is oxidized, (b) by following spectrophotometrically the appearance of reduced NADP in the reaction mixture at wavelengths of 340 or 366 mp, or (c) by observing the decolorization of a reducible dye such as methylene blue and brillant cresyl blue. 

Recently a colorimetric method for estimation of erythrocytic G-6-PDH was described (El). This procedure is based upon the interaction of phenazine methosulfate as electron carrier between NADPH2 formed in the reaction and dichloroindophenol, the rate of the reduction of the latter compound being followed at 620 mp. 

The observance of decolorization of a reducible dye may be suitable for a rapid screening test, but not for final examinations. Since the estimation of G-6-PDH activity may be necessary in laboratories where no photometer is available, both methods (b) and (c) mentioned above will be described. 

More recently the G-6-PDH method has been adapted especially for serum and hemolyzates (B15). 

For a quick decision, if G-6-PDH activity can be expected in a normal pattern, a screening test has been described (G7). The procedure is performed as follows 0.04 ml blood is mixed with 1.5 ml cold water for hemolysis then 0.4ml of tris buffer (pH 8.5, 0.74 M), 0.5ml brillant cresyl blue (IX 10-aM), 0.4ml H2O, 0.1ml NADP (0.1 mg), and 0.1 ml G-6-P solution (5 umoles) are added. After thorough mixing and sealing the mixture by the addition of about 1.0 ml mineral oil, the samples are incubated at 37°C and observed for decolorization. It should be noted whether or not decolorization occurs within 100 minutes of incubation ( = normal). 

All determinations of G-6-PDH activity should be made as soon as possible after the withdrawal of the blood sample. After standing for 24 hours, the activity was reported to have been reduced to 50% of the original (K7) storage conditions were not indicated. 

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