RESUMEN
Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.
Asunto(s)
Células de Sertoli/citología , Espermatogonias/citología , Nicho de Células Madre , Animales , Masculino , Células de Sertoli/metabolismo , Espermatogénesis , Espermatogonias/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
The monocyte chemoattractant protein 1 (MCP-1) belongs to the CC chemokine family and acts in the recruitment of C-C motif chemokine receptor 2 (CCR2)-positive immune cell types to inflammation sites. In testis, the MCP-1/CCR2 axis has been associated with the macrophage population's functional regulation, which presents significant functions supporting germ cell development. In this context, herein, we aimed to investigate the role of the chemokine receptor CCR2 in mice testicular environment and its impact on male sperm production. Using adult transgenic mice strain that had the CCR2 gene replaced by a red fluorescent protein gene, we showed a stage-dependent expression of CCR2 in type B spermatogonia and early primary spermatocytes. Several parameters related to sperm production were reduced in the absence of CCR2 protein, such as Sertoli cell efficiency, meiotic index, and overall yield of spermatogenesis. Daily sperm production decreased by almost 40%, and several damages in the seminiferous tubules were observed. Significant reduction in the expression of important genes related to the Sertoli cell function (Cnx43, Vim, Ocln, Spna2) and meiosis initiation (Stra8, Pcna, Prdm9, Msh5) occurred in comparison to controls. Also, the number of macrophages significantly decreased in the absence of CCR2 protein, along with a disturbance in Leydig cell steroidogenic activity. In summary, our results show that the non-activation of the MCP-1/CCR2 axis disturbs the testicular homeostasis, interfering in macrophage population, meiosis initiation, blood-testis barrier function, and androgen synthesis, leading to the malfunction of seminiferous tubules, decreased testosterone levels, defective sperm production, and lower fertility index.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Macrófagos/metabolismo , Receptores de Quimiocina/metabolismo , Espermatogénesis/fisiología , Testículo/fisiología , Animales , Femenino , Humanos , Masculino , RatonesRESUMEN
Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.
Asunto(s)
Escorpiones/citología , Espermatogonias/citología , Nicho de Células Madre , Tortugas/metabolismo , Animales , Biomarcadores/metabolismo , Forma de la Célula , Tamaño de la Célula , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Masculino , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Escorpiones/metabolismo , América del Sur , Espermatogonias/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Although several testicular alterations promoted by coronavirus infection have been demonstrated, the extent, causes, and players of testicular pathogenesis are not totally understood. The present study aimed to investigate the short-term effects on male fertility of intranasally administered murine hepatitis virus strain 3 (MHV-3), a member of the genus Betacoronavirus, which causes a severe systemic acute infection. This mouse model might be used as a in vivo prototype for investigating the impact of betacoronavirus on the endocrine and exocrine testicular functions with the advantage to be performed in a biosafety level 2 condition. Herein, we performed virological, histopathological, and molecular studies regarding the testicular spermatogenesis and the spermatic quality analyses in an MHV-3-infected C57BL/6 mice. The main outcomes showed that MHV-3 infects mouse testis and induces a testicular inflammatory state, impairing the steroidogenic pathway. The infection led to several alterations in the testicular parenchyma, such as: seminiferous epithelium sloughing, retention of residual bodies, germ cell apoptosis, alterations in intercellular junction proteins, and worse spermatogenic parameters. Moreover, the levels of plasmatic testosterone as well as the quality of sperm production reduced. Therefore, the present data suggest that the viral/inflammatory impairment of the steroidogenic pathway and the consequent imbalance of androgen levels is critical in testicular pathology, disturbing the SC barrier function and the germ cell differentiation. Our study is important for comprehending the effects of beta coronavirus infections on testis function in order to develop treatments that could prevent virus-mediated male infertility.
Asunto(s)
Ratones Endogámicos C57BL , Virus de la Hepatitis Murina , Espermatogénesis , Espermatozoides , Testículo , Animales , Masculino , Ratones , Testículo/virología , Testículo/patología , Testículo/inmunología , Espermatozoides/virología , Espermatozoides/inmunología , Espermatozoides/patología , Modelos Animales de Enfermedad , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Infertilidad Masculina/virología , Infertilidad Masculina/inmunología , Infertilidad Masculina/patología , Infertilidad Masculina/etiología , Testosterona/sangre , HumanosRESUMEN
Acute promyelocytic leukemia (APL) is usually associated with a favorable outcome, but about 10% of patients tend to relapse. The genetic hallmark of APL is a balanced translocation involving chromosomes 15 and 17, and the PML-RARa gene fusion is found in more than 90% of these cases. Other chromosomal abnormalities are commonly found in APL, but their clinical significance has yet to be determined. Here we report a case of childhood APL that was studied by conventional cytogenetics along with molecular cytogenetic techniques. The patient showed a complex karyotype with an unusual cytogenetic rearrangement originating from two different abnormalities in a single chromosome 6. Our case is an exceptional example of a cryptic cytogenetic anomaly in APL and underscores the importance of detailed genetic characterization.
Asunto(s)
Cromosomas Humanos Par 6 , Reordenamiento Génico , Leucemia Promielocítica Aguda/genética , Translocación Genética/genética , Niño , Bandeo Cromosómico , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Humanos , Hibridación Fluorescente in Situ , MasculinoRESUMEN
Constitutional genomic imbalances are known to cause malformations, disabilities, neurodevelopmental delay, and dysmorphia and can lead to dysfunctions in the cell cycle. In extremely rare genetic conditions such as small supernumerary marker chromosomes (sSMC), it is important to understand the cellular consequences of this extra marker, as well the factors that contribute to their maintenance or elimination through successive cell cycles and phenotypic impact. The study of chromosomal mosaicism provides a natural model to characterize the effect of aneuploidy on genome stability and compare cells with the same genetic background and environment exposure, but differing in the presence of sSMC. Here, we report the functional characterization of different cell lines from two familial patients with mosaic sSMC derived from chromosome 12. We performed studies of proliferation dynamics, stability, and variability of these cells using fluorescent in situ hybridization (FISH), sister chromatid exchanges (SCE), and conventional staining. We also quantified the telomere-related genomic instability of sSMC cells using 3D telomeric profile analysis by quantitative-FISH. sSMC cells exhibited differences in the cell cycle dynamics compared to normal cells. First, the sSMC cells exhibited lower proliferation index and higher frequency of SCE than normal cells, associated with a higher level of chromosomal instability. Second, sSMC cells exhibited more telomeric-related genomic instability. Lastly, the differences of sSMC cells distribution among tissues could explain different phenotypic repercussions observed in patients. These results will help in our understanding of the sSMC stability, maintenance during cell cycle, and the cell cycle variables involved in the different phenotypic manifestations.
Asunto(s)
Cromosomas Humanos Par 12 , Mosaicismo , Padre , Marcadores Genéticos/genética , Inestabilidad Genómica/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Núcleo FamiliarRESUMEN
Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.
Asunto(s)
Zarigüeyas/fisiología , Espermatogénesis/fisiología , Animales , Masculino , Epitelio Seminífero/fisiología , Especificidad de la Especie , Espermatogénesis/efectos de los fármacos , Timidina/farmacologíaRESUMEN
Constitutional genomic imbalances are known to cause malformations, disabilities, neurodevelopmental delay, and dysmorphia and can lead to dysfunctions in the cell cycle. In extremely rare genetic conditions such as small supernumerary marker chromosomes (sSMC), it is important to understand the cellular consequences of this extra marker, as well the factors that contribute to their maintenance or elimination through successive cell cycles and phenotypic impact. The study of chromosomal mosaicism provides a natural model to characterize the effect of aneuploidy on genome stability and compare cells with the same genetic background and environment exposure, but differing in the presence of sSMC. Here, we report the functional characterization of different cell lines from two familial patients with mosaic sSMC derived from chromosome 12. We performed studies of proliferation dynamics, stability, and variability of these cells using fluorescent in situ hybridization (FISH), sister chromatid exchanges (SCE), and conventional staining. We also quantified the telomere-related genomic instability of sSMC cells using 3D telomeric profile analysis by quantitative-FISH. sSMC cells exhibited differences in the cell cycle dynamics compared to normal cells. First, the sSMC cells exhibited lower proliferation index and higher frequency of SCE than normal cells, associated with a higher level of chromosomal instability. Second, sSMC cells exhibited more telomeric-related genomic instability. Lastly, the differences of sSMC cells distribution among tissues could explain different phenotypic repercussions observed in patients. These results will help in our understanding of the sSMC stability, maintenance during cell cycle, and the cell cycle variables involved in the different phenotypic manifestations.
RESUMEN
Sertoli cells (SCs) play a crucial role in testis differentiation, development and function, determining the magnitude of sperm production in sexually mature animals. For over 40 years, it has been considered that these key testis somatic cells stop dividing during early pre-pubertal phase, between around 10 to 20 days after birth respectively in mice and rats, being after that under physiological conditions a stable and terminally differentiated population. However, evidences from the literature are challenging this dogma. In the present study, using several important functional markers (Ki-67, BrdU, p27, GATA-4, Androgen Receptor), we investigated the SC differentiation status in 36 days old and adult Wistar rats, focusing mainly in the transition region (TR) between the seminiferous tubules (ST) and the rete testis. Our results showed that SCs in TR remain undifferentiated for a longer period and, although at a lesser degree, even in adult rats proliferating SCs were observed in this region. Therefore, these findings suggest that, different from the other ST regions investigated, SCs residing in the TR exhibit a distinct functional phenotype. These undifferentiated SCs may compose a subpopulation of SC progenitors that reside in a specific microenvironment capable of growing the ST length if needed from this particular testis region. Moreover, our findings demonstrate an important aspect of testis function in mammals and opens new venues for other experimental approaches to the investigation of SC physiology, spermatogenesis progression and testis growth. Besides that, the TR may represent an important site for pathophysiological investigations and cellular interactions in the testis.
Asunto(s)
Envejecimiento/fisiología , Red Testicular/citología , Túbulos Seminíferos/citología , Células de Sertoli/citología , Células de Sertoli/metabolismo , Animales , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciación Celular , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA4/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Ratas Wistar , Receptores Androgénicos/metabolismo , Maduración SexualRESUMEN
Identification of inactive prorenin in the kidney has been difficult due to rapid proteolytic conversion of the inactive zymogen to its active form in the tissue or during homogenization and purification. Immunochemical methods, Western blotting, direct radioimmunoassay, and immunoaffinity chromatography were used to isolate and identify rat kidney renin and prorenin and to determine their molecular weights without complete purification. Antisera to pure rat renin were raised in rabbits. A specific reaction between the antisera and rat renin was demonstrated by double immunodiffusion, inhibition of enzyme activity, and competitive radioimmunoassay. The anti-rat renin IgG did not cross-react with purified human renin or rat spleen or kidney cathepsin D. The IgG showed binding affinity to both inactive renin as well as active enzyme. A combination of affinity chromatographies consisting of pepstatin-Sepharose, IgG-Sepharose, and Affi-Gel Blue permitted rapid and complete separation of inactive renin from active renin in rat kidney extract. Neither inactive nor active renin preparations exhibited aspartyl protease activity on hemoglobin used as substrate. The apparent molecular weight of inactive renin was estimated as 50,000 by gel filtration. Electrophoresis of partially purified inactive renin in sodium dodecyl sulfate (SDS) polyacrylamide gel followed by transblotting of proteins to a nitrocellulose sheet and immunochemical staining with anti-renin IgG showed a single protein band with a molecular weight of 48,000. Activation of inactive renin by trypsin was accompanied by the reduction of the 48,000-dalton native protein to a 39,000-dalton protein as determined by the SDS polyacrylamide gel electrophoresis and the transblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Riñón/enzimología , Renina/aislamiento & purificación , Animales , Anticuerpos/inmunología , Catepsina D/inmunología , Catepsina D/aislamiento & purificación , Catepsina D/metabolismo , Cromatografía/métodos , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Precursores Enzimáticos/inmunología , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Humanos , Inmunoglobulina G/inmunología , Riñón/inmunología , Ratones , Peso Molecular , Radioinmunoensayo , Ratas , Renina/inmunología , Renina/metabolismo , Bazo/metabolismoRESUMEN
Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.
Asunto(s)
Calicreínas/farmacología , Quininógenos/fisiología , Contracción Uterina/efectos de los fármacos , Animales , Aprotinina/farmacología , Bradiquinina/antagonistas & inhibidores , Bradiquinina/farmacología , Femenino , Técnicas In Vitro , Calicreínas/antagonistas & inhibidores , Cininas/metabolismo , Ratas , Ratas Endogámicas , Glándula Submandibular/análisisRESUMEN
Specific high-titer antibodies to rat kidney renin were raised using pure rat kidney renin conjugated with tetanus toxoid as antigen. The titers of two antisera for 50% inhibition of rat kidney renin activity were 80 000 and 700 000, respectively. The antibody inhibited the enzyme activities of hog kidney renin and mouse submaxillary gland renin at concentrations 1 000 times higher than required for rat kidney renin but did not inhibit human renal renin or rat kidney cathepsin D. The antibody can be used for characterizing the physiological and pathophysiological role of renin in blood pressure regulation, for the histochemical localization of renin in rat tissues and for distinguishing specific renin activity from the nonspecific renin-like activity of cathepsin D.
Asunto(s)
Anticuerpos/inmunología , Renina/inmunología , Animales , Formación de Anticuerpos , Catepsina D/aislamiento & purificación , Catepsina D/metabolismo , Humanos , Sueros Inmunes/inmunología , Inmunización , Riñón/enzimología , Conejos , Ratas , Renina/metabolismo , Porcinos , Toxoide Tetánico/inmunologíaRESUMEN
Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.
Asunto(s)
Compuestos de Anilina/farmacología , Benzamidinas/farmacología , Compuestos Cromogénicos/química , Oligopéptidos/metabolismo , Calicreínas de Tejido/antagonistas & inhibidores , Amidohidrolasas/química , Sitios de Unión , Humanos , Hidrólisis , Modelos Lineales , Proteínas de Plantas/farmacología , Calicreínas de Tejido/química , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.
Asunto(s)
Aprotinina/farmacocinética , Calicreínas/orina , Inhibidores de Tripsina/farmacocinética , Aprotinina/metabolismo , Sitios de Unión , Unión Competitiva , Humanos , Calicreínas/antagonistas & inhibidores , Masculino , Peso Molecular , Análisis de Regresión , Inhibidores de Tripsina/metabolismoRESUMEN
Hydrolysis of seven N alpha-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.
Asunto(s)
Arginina/análogos & derivados , Calicreínas/farmacocinética , Animales , Arginina/metabolismo , Hidrólisis , Calicreínas/aislamiento & purificación , Calicreínas/orina , Ratas , Ciclo del SustratoRESUMEN
A kininase has been purified from male human urine which splits the C-terminal arginyl residue from bradykinin, but does not split hippuryl-L-arginine or converts Angiotensin I into Angiotensin II. It contains cadmium ion in its active center, and has a molecular weight of 210,000 by ultracentrifugation.
Asunto(s)
Carboxipeptidasas/orina , Lisina Carboxipeptidasa/orina , Ácido Edético/farmacología , Humanos , Cinética , Lisina Carboxipeptidasa/aislamiento & purificación , Masculino , Peso Molecular , Especificidad por SustratoRESUMEN
A simple, large scale process for the purification of human urinary kallikrein is described which is based upon concentration by reverse osmosis, ammonium sulfate precipitation, and gel filtration on a column of Sephadex-G-150. The yield in protein is higher than any reported in the literature to this date; the purified enzyme seems to be identical to that reported by Figueiredo and Mares-Guia at the Kinin-Symposium in Paris, 1978, based on the procedure of Hial, et al., (1974).