Histochemical and biochemical studies of the kinetics of non-specific acid phosphatase in rat kidney.

Kinetic characteristics of non-specific acid phosphatase (orthophosphoric monoester phosphohydrolase, E.C.3.1.3.2.) from rat kidney were determined fluorometrically using 4-methylumbelliferyl phosphate as the substrate. Kinetic characteristics measured by similar methods both histochemically in cryostat sections and biochemically in tissue extracts were compared. Histochemical and biochemical methods gave essentially similar results in respect of Michaelis-Menten constants (Km), pH optima, effect of fluoride inhibition and the effect of changes in incubation temperatures in the range 10 degrees C to 37 degrees C. This confirms the validity of both methods, and also gives greater confidence that the enzyme in vitro closely approximates the properties of the enzyme as it functions in vivo.

An improved method for staining cells of the endocrine polypeptide (APUD) series by masked metachromasia: application of the principle of 'fixation by excluded volume'.

Masked metachromasia is demonstrated by staining with a metachromatic basic dye, after acid hydrolysis of suitably fixed tissue. We report that the addition of 20% Carbowax 20M (an inert polyer, mol. wt. about 20,000) to the hydrolysis mixture improved the reaction. The improved method gives increased metachromasia , greater tolerance to variations in hydrolysis conditions, and demonstrates a greater proportion of cells--presumably due to a lower threshold of sensitivity. Lower molecular weight polymers (Carbowax 1000, Carbowax 6000) are less effective.

Storage granules of thyroid C cells in the dog: a cytochemical and ultrastructural study, in relation to the masked metachromasia reaction.

Masked metachromasia can be demonstrated in thyroid C cells, and other cells of the APUD series, by staining with a metachromatic basic dye after hydrolysis of suitably fixed tissue. The reaction is thought to be due to the presence of polypeptides with a high concentration of side-chain acidic groups. Since most APUD cells possess storage granules, presumed to contain a polypeptide hormone, it has been assumed that the masked metachromasia reaction gives information concerning the contents of these granules. However, there has been an increasing suspicion that the reaction might actually be due to the membrane bounding these granules, rather than to the contents. We have examined, cytochemically and ultrastructurally, dog thyroid tissue which has been subjected to fixation and hydrolysis as in the usual method for masked metachromasia. We found that the membrane surrounding the C cell granules is removed by hydrolysis, confirming the hypothesis that the reaction is due to the contents (hormone and/or matrix)rather than to the membrane. Tissues were fixed in an aqueous mixture containing glutaraldehyde (6 25% v/v), picric acid (three-quarters saturation) and sodium acetate (I% W/V)adjusted to PH 7 with sodium hydroxide. This was found to be a very satisfactory fixative for electron microscopy Some morphological details of C cells were noted, such as the richness of desmosomes between C cells in this species, and frequent direct contact with the colloid.