Simplified control experiments in the histochemical study of coenzyme-linked dehydrogenases

Summary

Different experiments concerning some of the most critical steps in the histochemical procedure for coenzyme-linked dehydrogenases (fixation procedure, cryoprotection, osmolar protection, substantivity of formazans, “nothing dehydrogenase” reaction, diffusion of enzyme, rediffusion of reduced coenzyme and/or reduced phenazine methosulphate) were carried out in order to improve or introduce simplified control methods by which the in situ localization of enzyme activity can be achieved without the need of expensive equipments.

As a test-object glucose-6-phosphate dehydrogenase was used.

Brief (5 min) prefixation of tissue (liver) at 0–4° C with 1% buffered (pH=7.2) methanolfree formaldehyde (from paraformaldehyde) gave excellent preservation of morphology during the procedures of freezing, cutting and incubation and caused no inhibition of G6PDH. With the named fixation no improvement was obtained by the simultaneous use of cryoprotection (DMSO) or osmolar protection (sucrose). Finally, the fixation caused an enhancement of Nitro BT penetration into the tissue as well as of formazan substantivity. On the other hand, the brief fixation did not abolish the diffusion of enzyme (proved by different methods) and of reduced coenzyme or reduced phenazine methosulphate.

In a conventional aqueous incubation medium as well as in a gel incubation medium (PVA, grade Bo5/140) the rate of diffusion of reduced coenzyme and/or reduced phenazine methosulphate was investigated by using a special double-section incubation method. The concentrations of Nitro BT, NADP and PMS were balanced against each other and it was concluded that by using a gel medium containing 0.5 mg/ml Nitro BT, 0.1 mg/ml NADP and 0.003 mg/ml PMS, the in situ localization of G6PDH activity could be achieved at the cellular level with an incubation time not exceeding 10 min. With the incubation time mentioned, the “nothing dehydrogenase” reaction was out of the question. The sensibility of the double-section incubation method is discussed and provided that the dehydrogenase in question contains sulphydryl groups in the active enzyme centre, the method seems to exhibit a sufficient high level of sensitivity in the control of the diffusion of the different components operative in the histochemical dehydrogenase procedure.

The recording of the incubation time needed for the appearance of the two formazans (red and blue) is recommended in order to follow the enzymatic reaction rate and the effect of different procedures (fixation, solvents added etc.) as well as the rates of “nothing dehydrogenase” reaction, diffusion of reduced coenzyme and/or reduced PMS.