ULTRASTRUCTURAL LOCALIZATION OF ALKALINE PHOSPHATASE ACTIVITY IN THE ABSORBING CELLS OF THE DUODENUM OF MOUSEHUGON, J.; BORGERS, M.
doi: 10.1177/14.9.629pmid: 5339389
A direct lead method for the visualization of alkaline phosphatase activity at pH 9 was used in a study on the absorptive cells of the duodenum of the mouse. Lead phosphate precipitate was observed in several structures after a short incubation of glutaraldehyde-fixed frozen sections. On microvilli and in vacuoles below the terminal web, the reaction was intense and persisted even after osmic acid fixation. In the Golgi zone, in the smooth reticulum cisternae and in the dense bodies, the reaction was prominent. In these sites, the deposits were not observed after osmic acid fixation or after short incubation in media containing substrates other than β-glycerophosphate or α-naphthylphosphate.
THE DEMONSTRATION OF ACID HYDROLASE, THERMOSTABLE REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE TETRAZOLIUM REDUCTASE AND PEROXIDASE ACTIVITIES IN HUMAN LIPOFUSCIN PIGMENT GRANULESGOLDFISCHER, SIDNEY; VILLAVERDE, HUMBERTO; FORSCHIRM, REGINA
doi: 10.1177/14.9.641pmid: 4165860
The characterization of human lipofuscin pigment granules as lysosomes is strengthened by the cytochemical demonstration of β-glucuronidase and glucosaminidase activities in hepatocyte pigment granules which also have acid phosphatase activity. Lysosomes of parenchymal cells and macrophages are seen to differ in their content of cytochemically demonstrable hydrolase activities. Reduced diphosphopyridine nucleotide tetrazolium reductase and peroxidase activities that are remarkably resistant to heat (90°C for 15 min) and prolonged fixation are also visualized in lipofuscin granules. It is suggested that these oxidative activities reflect the presence of heme compounds that accumulate during the degradation of cytoplasmic constituents in autophagic vacuoles or dense bodies. The presence of metabolites with peroxidase activity within lysosomes may be responsible for the oxidation of melanin precursors to melanin and unsaturated fat to the alcohol-insoluble lipid of lipofuscin pigment. Cytoplasmic iron (hemosiderin) that is stained by Perls' ferrocyanide or Quinke's sulfide procedures is also visualized by incubating sections for peroxidase activity in Karnovsky's 3,3'-diaminobenzidine medium. This activity disappears following the extraction of stainable iron in Lillie's dithionite medium. However, the heme-associated peroxidase activity is unaffected.
CHARACTERIZATION OF ENZYMES HYDROLYZING ACYL NAPHTHYLAMIDES III. ROLE OF KYNURENINE FORMAMIDASEHOPSU, V. K.; SANTTI, R.; GLENNER, G. G.
doi: 10.1177/14.9.653pmid: 4165861
An enzyme fraction in guinea pig liver catalyzing the hydrolysis of chloroacetyl α-naphthylamide was separated by gel filtration and demonstrated to hydrolyze N-formyl-l-kynurenine. On the basis of relative substrate hydrolysis rates, the enzymes in this peak were shown to have the characteristics of kynurenine formamidase, an enzyme catalyzing the transformation of N-formyl-l-kynurenine to l-kynurenine and formic acid. The histochemical localization in fixed tissue of the hydrolysis of chloroacetyl α-naphthylamide can be almost totally ascribed to this enzyme and, thereby, indicates tissue sites of tryptophan catabolism.
SUBFRACTIONS OF LACTATE DEHYDROGENASE OF THE RATBUTA, JANET L.; CONKLIN, JAMES L.; DEWEY, MAYNARD M.
doi: 10.1177/14.9.658pmid: 5971219
A survey of the lactate dehydrogenase (LDH) isozymes of 20 organs and tissues of the rat revealed a total of nine fractions exhibiting LDH activity after starch gel electrophoresis. Of the nine LDH fractions, eight were found in testis and lymph node, while the ninth fraction was unique to kidney. The occurrence of LDH fractions in excess of the usual five LDH isozymes is accounted for by two alternate proposals. One proposal is an extension of the present tetramer postulate of LDH structure, while the other is a consideration of the significance of heterozygosity in the albino rats employed in the study. Both hypotheses are plausible and should be considered in future studies of isozymes.
TISSUE FIXATION BY DIAZONIUM SALTS OF A PARTICLE-BOUND RAT KIDNEY AMINOPEPTIDASENAGATSU, I.; MASON, S.; GLENNER, G. G.
doi: 10.1177/14.9.663pmid: 5971220
Several rapid coupling diazonium salts (fast garnet GBC, fast red B and fast blue R) have been shown to cause the retention by fixation in tissue sections of the buffer-extractable portion of a particle-bound aminopeptidase catalyzing the hydrolysis of l-leucyl β-naphthylamide. The enzyme retained by these diazonium salts is identical to a cobalt-activated aminopeptidase which remains bound to tissues after buffer washing, but is distinct from a soluble sulfhydryl-dependent aminoptipedase extracted from tissues during incubation. No one of the three diazonium salts tested at equivalent coupling concentrations produced more retention of enzyme in tissue section than another, while the surfactants and salts present in commercial garnet GBC had no discernible effect in preventing enzyme extraction.
A HISTOCHEMICAL AND BIOCHEMICAL STUDY OF NUCLEAR ADENOSINE TRIPHOSPHATE HYDROLYSIS IN EMBRYO HEARTKLEIN, RICHARD L.
doi: 10.1177/14.9.669pmid: 4165862
A correlative histochemical study using cryostat sections and a biochemical study using isolated nuclei were undertaken to characterize nuclear adenosine triphosphate (ATP) hydrolysis in embryonic myocardium of the chick. Qualitatively, the data give an unequivocal positive correlation between the two studies except for minor sensitivity differences. A variety of criteria establish the enzymatic nature of the nuclear ATP hydrolysis and affirm the presence of at least one ATP phosphohydrolase. Enzyme activity is probably localized both at the nuclear surface and in the nucleoplasm. Mg++-activated ATP phosphohydrolase is predominant over the Ca++-activated enzyme, and the former is stimulated by the addition of NaCl. The characteristics of the enzyme activity and the combined methods employed eliminate the possibility that the observed activity is due to artifact or to contamination by other cell fractions.