RESUMO
The ultracytochemical localization of amine oxidase (AO) activity is demonstrated with a new substrate, p-N,N-dimethylamino-beta-phenethylamine (DAPA). DAPA was designed to yield a stronger reducing agent on oxidation by monoamine oxidase (MAO) than is obtained from the MAO substrate, tryptamine, upon oxidation. Thus MAO and possibly other oxidase(s) can be demonstrated with DAPA and the tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). The latter is a nonosmiophilic tetrazolium salt which is reduced to an osmiophilic formazan. In addition, DAPA itself demonstrates AO activity ultracytochemically with and without BSPT. We speculate that either oxidative polymerization of DAPA or Schiff's base formation with protein after aldehyde formation is responsible for the latter reaction, which is made permanent for ultracytochemical localization by osmication at a later step. DAPA oxidation reaction products are demonstrated in guinea pig kidney, specifically in the endoplasmic reticulum, nuclear envelope and mitochondrial outer compartments and cristae. Differences in reaction product characteristics and localization in relation to formaldehyde fixation and the localization of reaction product in mitochondrial cristae, as well as outer compartments, suggest that DAPA oxidation is mediated through one or more MAOs and possible other oxidases.
Assuntos
Monoaminoxidase/análise , Fenetilaminas , Animais , Cobaias , Histocitoquímica , Rim/enzimologia , Rim/ultraestrutura , Masculino , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , OxirreduçãoRESUMO
A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.
Assuntos
Formaldeído/farmacologia , Oxirredutases/metabolismo , Animais , Histocitoquímica , Indicadores e Reagentes , Rim/enzimologia , Rim/ultraestrutura , Fígado/enzimologia , Fígado/ultraestrutura , Masculino , Músculos/enzimologia , Músculos/ultraestrutura , Miocárdio/enzimologia , Miocárdio/ultraestrutura , Nitrocompostos , Próstata/enzimologia , Próstata/ultraestrutura , Ratos , Sais de TetrazólioRESUMO
By taking advantage of the structural requirements of the substrates for prostatic acid phosphatase (PAP), which consist of steric hindrance and the presence of basic nitrogen in the molecule, potential cytotoxic agents (spindle poisons) are being designed that will become enzyme activated specifically by PAP. Colchicine has been converted to colchiceinamides of substituted ethanolamines and o-phosphoethanolamines. The rate of hydrolysis of the latter by human prostatic tissue as compared to the rate of hydrolysis by human kidneys (P/K ratio) is given and indicates a significant degree of specificity for PAP. Some preliminary toxicity data in mice are also given. New thiocolchicine derivatives with phosphates on ring B are also being prepared for study and some preliminary toxicity data are given. The observation in biochemical experiments that phosphorylcholine is a very specific substrate for PAP has led us to develop specific cytochemical methods for PAP for both light and electron microscopy. Preliminary observations are given and good evidence is provided that PAP is not a lysosomal enzyme, unlike other acid phosphatases. Furthermore, PAP is to other acid phosphatases what the cholinesterases are to other esterases. Since the acid phosphatase that is able to hydrolyze phosphorylcholine is characteristic of prostatic epithelium, this is the acid phosphatase that is referred to be the designation of PAP. Other acid phosphatases (both lysosomal and nonlysosomal) in prostatic epithelial cells are not demonstrated by this substrate and hence are not included in this designation.
Assuntos
Fosfatase Ácida/farmacologia , Alquilantes/farmacologia , Próstata/enzimologia , Animais , Cães , Ativação Enzimática , Radicais Livres , Haplorrinos , Histocitoquímica , Humanos , Hidrólise , Córtex Renal/enzimologia , Masculino , Camundongos , Microscopia Eletrônica , Nitrogênio/metabolismo , Papio , Fosforilcolina , Próstata/citologia , Coelhos , RatosAssuntos
Cloroplastos/enzimologia , Miocárdio/enzimologia , Osmio , Succinato Desidrogenase/análise , Sais de Tetrazólio/síntese química , Animais , Histocitoquímica , Técnicas In Vitro , Microscopia Eletrônica , Músculos/citologia , Músculos/enzimologia , Miocárdio/citologia , Fotoquímica , Células Vegetais , Ratos , Solubilidade , Coloração e RotulagemAssuntos
Colinesterases/análise , Compostos de Diazônio , Indóis , Junção Neuromuscular/enzimologia , Tolueno , Animais , Compostos Azo , Éteres , Histocitoquímica , Hidrólise , Músculos Intercostais/anatomia & histologia , Músculos Intercostais/citologia , Microscopia , Microscopia Eletrônica , Polímeros , Ratos , Coloração e Rotulagem , Sulfetos , Sinapses/enzimologia , Temperatura , Fatores de TempoAssuntos
Oxirredutases/análise , Coloração e Rotulagem , Estirenos , Sais de Tetrazólio , Animais , Di-Hidrolipoamida Desidrogenase/análise , Dimetil Sulfóxido , Complexo IV da Cadeia de Transporte de Elétrons/análise , Retículo Endoplasmático/enzimologia , Formaldeído , Histocitoquímica , L-Lactato Desidrogenase/análise , Métodos , Microscopia Eletrônica , Mitocôndrias Musculares/enzimologia , Músculos/citologia , Miocárdio/enzimologia , Osmio , Ratos , Estirenos/síntese química , Succinato Desidrogenase/análise , Sais de Tetrazólio/síntese química , VibraçãoAssuntos
Acetilcolinesterase/análise , Compostos de Diazônio , Osmio , Polímeros , Acetatos , Compostos de Anilina/análise , Animais , Compostos Azo/análise , Fenômenos Químicos , Química , Colinesterases/análise , Temperatura Baixa , Diafragma/análise , Esterases/análise , Éteres , Histocitoquímica , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Músculos Intercostais/citologia , Substâncias Macromoleculares/análise , Métodos , Microscopia Eletrônica , Junção Neuromuscular/citologia , Ratos , Coloração e Rotulagem , Sulfetos/análiseAssuntos
Aciltransferases/metabolismo , Aminopeptidases/metabolismo , Compostos Azo , Compostos de Diazônio , Retículo Endoplasmático/enzimologia , Rim/enzimologia , Pâncreas/enzimologia , Animais , Histocitoquímica , Rim/citologia , Masculino , Microscopia Eletrônica , Pâncreas/citologia , Piridazinas , RatosAssuntos
Compostos de Anilina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , p-Dimetilaminoazobenzeno/farmacologia , Compostos de Anilina/síntese química , Animais , Retículo Endoplasmático , Fígado/enzimologia , Mitocôndrias , Mitocôndrias Hepáticas , Miocárdio/enzimologia , RatosAssuntos
Compostos de Anilina , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Peroxidases/metabolismo , Animais , Fenômenos Químicos , Química , Histocitoquímica , Técnicas In Vitro , Indicadores e Reagentes , Rim/enzimologia , Fígado/enzimologia , Miocárdio/enzimologia , p-DimetilaminoazobenzenoAssuntos
Compostos de Anilina , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Histocitoquímica , Indicadores e Reagentes , Naftalenos , Anilidas , Animais , Ferrocianetos , Indicadores e Reagentes/síntese química , Rim/enzimologia , Fígado/enzimologia , Masculino , Microscopia Eletrônica , Miocárdio/enzimologia , Nitrobenzenos , Peroxidases , Piperazinas , Polímeros , Ratos , TioureiaRESUMO
A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).