Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.     

Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes

Synopsis Fixation under improper conditions ofin vitro cultivated cells results in an extensive diffusion of the lysosomal enzyme acid phosphatase because of the influence of a low effective osmotic pressure. In the present investigation, advantage was taken of this predictable diffusion in order to establish whether or not leakage of acid phosphatase could take place through ultrastructurally intact lysosomal membranes.In order to reveal small holes in the lysosomal membranes, secondary lysosomes were labelled with thorium dioxide particles, which were presumed to appear free in the cell sap if ruptures in the membranes larger than about Ioo Å were created.The experiments revealed that following the fixation ofin vitro cultivated human glia cells under improper conditions, mitochondria and ground cytoplasm show considerable swelling artifacts, while secondary lysosomes appear to be essentially unaffected. The lysosomes, nevertheless, apparently lost most of their content of acid phosphatase, as judged from enzyme cytochemical studies. These findings indicate that leakage of acid phosphatase from ultrastructurally intact lysosomes is possible.     

Cytochemical demonstration of acid phosphatase, trimetaphosphatase and basic protein in rat peritoneal mast cells during 48/80 induced exocytosis

Acid phosphatase (AcP-A), trimetaphosphatase (TmP-A) activities and basic protein reaction were cytochemically studied in rat peritoneal mast cells 15 minutes after stimulation by compound 48/80. The AcPase reaction was positive in slightly altered granules, but negative in those more intensely altered, and also in unaltered granules. The TmP-A reaction was negative in altered granules and positive in a few unaltered granules. These results suggest that mast cells have two populations of granules, one, comprising most granules, is AcP-A positive and is exocytosed. The other, smaller, is TmP-A positive, and is not exocytosed. Intact granules gave a strong positive reaction with amoniacal silver nitrate (AS), which detects basic protein. This reaction decreased in intensity with increasing granule alteration.     

Cytochemical localization of acid phosphatase in striated muscle

Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.     

Cytochemical localization of acid phosphatase in striated muscle

Summary Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.Supported in part by grants AI 17945 and HL 17747 from the United States Public Health Service, National Institutes of Health     

Use of osmium-ferrocyanide treatment for improved lysosomal acid trimetaphosphatase reaction and subcellular detail in thyroid follicular cells

The histochemical reaction for acid trimetaphosphatase in addition to secondary tissue treatment with an osmium-ferrocyanide mixture was used to study lysosomes and phagolysosomes in the mouse thyroid gland. The osmium-ferrocyanide postfixation enhanced reaction product localization, reduced diffuse reaction, and improved membrane contrast. In addition, the ultrathin tissue sections did not require heavy metal staining, thus eliminating potential stain artifacts due to precipitation. In view of the improved tissue preservation and enzyme localization, it is suggested that osmium-ferrocyanide postfixation be used after the acid trimetaphosphatase method.     

Molecular cloning of the mouse lysosomal acid phosphatase

The mouse cDNA for lysosomal acid phosphatase was cloned. The deduced amino-acid sequence shows 89 and 96% identity with that of the human and rat enzyme, respectively. Namely all residues known to be important for the structure, catalytic activity and transport of lysosomal acid phosphatase are conserved among the three species.     

Targeting of lysosomal acid phosphatase with altered carbohydrate

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes, where it is converted into a soluble protein by a limited proteolysis (Waheed et al., 1988, EMBO J. 7, 2351-2358). Transport of human lysosomal acid phosphatase in heterologous BHK-21 cells was examined under conditions that impair mannose-6-phosphate receptor-dependent transport, N-glycosylation or processing of N-linked oligosaccharides. Targeting of lysosomal acid phosphatase to lysosomes was neither affected by antibodies blocking the mannose-6-phosphate/IGF II receptor, nor by NH4Cl, which inhibited the mannose-6-phosphate receptor-dependent targeting of soluble lysosomal enzymes. 1-Deoxynojirimycin, 1-deoxymannojirimycin and swainsonine inhibited processing of N-linked oligosaccharides in lysosomal acid phosphatase without significantly affecting its transport. Tunicamycin inhibited N-glycosylation of lysosomal acid phosphatase. The non-glycosylated lysosomal acid phosphatase polypeptides accumulated within light membranes and were not transported to dense lysosomes. These results indicate that transport of lysosomal acid phosphatase is independent of mannose-6-phosphate receptors, does not involve an acid pH-dependent step and does not require processing of N-linked oligosaccharides. N-glycosylation appears to be necessary to achieve a transport competent form of lysosomal acid phosphatase.     

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo

Summary The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulumlysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.     

Cytochemical and biochemical determination of acid phosphatase activity in yeasts]

Optimal conditions of the cytochemical assay for acid phosphatase in protoplasts and whole cells of S. cerevisiae have been described. Dimethyl sulfoxide was used to increase the permeability of the yeast cell envelope. In the yeast cells, grown up to the end of the exponential phase, acid phosphatase is shown to be located mainly in the central vacuole and on the cell envelope surface. A considerable activity of acid phosphatase is demonstrable on the surface of the plasma membrane and within adjacent vesicles that represent, presumably, part of the endoplasmic reticulum. Acid phosphatase can be considered as a marker enzyme for yeast cell vacuoles.     

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5′ promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5′ up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.