Histochemical demonstration of human liver hexokinase, low Km hexokinase, Km for glucose 10-4M or less (HK), and glucokinase, Km for glucose 10-1M(GK), was described in control and liver disease patients. The method was essentially an application of kinetic assay system of HK and GK established by Viñuela et al. The activities of HK and GK were demonstrated as a dark blue deposit of formazan, which produced by the reduction of nitro blue tetrazolium (nitro-BT) with NADPH, an intermediate in the HK reaction coupled to the oxidation of glucose-6-phosphate (G6P). The specificity of present method was indicated by the lack of color development in the absence of either ATP or glucose. The intensity of formazan deposition appeared to represent the activity of HK and GK in the human liver tissue. HK was demonstrated when stained with 0.5mM glucose concentration and HK and GK was demonstrated with 100mM glucose concentration.
Aminopeptidase activity contained in healing fractures was studied histochemically in fibrous, caritlaginous and osseous callus from rat long bones. The substrates used consisted of amino acid β-naphthylamine compounds, i.e., L-alanine, L-leucine, L-arginine, DL-methionine, L-proline and L-alanine-4-methoxy compounds. The aminopeptidase was intensely confined to fibrous and cartilaginous callus cells and was less reactive in ossous callus. Osteoclasts and chondroclasts were devoid of the enzymatic activity irrespective of high amounts of acid phosphatase and succinate dehydrogenase. No significant differences for the aminopeptidase activity were observed in callous tissue when several amino acids compounds were used as substrate.
The morphogenesis of peroxisomes was investigated ultrastructurally in hepatocytes of normal mice and mice given simfibrate (hypolipidemic agent) in a daily dose of 1, 000mg/kg for 1 to 14 days. Continuity between the rough endoplasmic reticulum and peroxisomes of different sizes was most frequently observed. A characteristic loop- or hook-shaped smooth endoplasmic reticulum was often visualized as a continuity with peroxisomes or with the rough endoplasmic reticulum. These observations lead to the suggestion that the loop- or hook-shaped smooth endoplasmic reticulum may play an important role in the initial stage of formation of mouse peroxisomes.