RESUMEN
Cisplatin is a widely used chemotherapeutic agent whose dose-limiting side effects include ototoxicity and nephrotoxicity. Recent evidence indicates that cisplatin induces the expression of a novel protein, kidney injury molecule-1, in the renal proximal tubular epithelium to aid in regeneration. In this study, we determined whether kidney injury molecule-1 is expressed in the cochlea and is induced by cisplatin. Using reverse transcriptase polymerase chain reaction techniques, we have now identified kidney injury molecule-1 in the rat cochlea and in three different mouse transformed hair cell lines. Administration of cisplatin to rats produced hearing loss and induced kidney injury molecule-1 mRNA in the rat cochlea. Pretreatment of rats with lipoic acid, a scavenger of reactive oxygen species, significantly reduced cisplatin-induced hearing loss and kidney injury molecule-1 expression. Cisplatin also increased the expression of cochlear NOX3 mRNA, a member of the superoxide generating NADPH oxidase family of proteins recently identified in the cochlea, inhibition of which decreased kidney injury molecule-1 expression. Polymerase chain reaction performed on different regions of the cochlea indicated the presence of kidney injury molecule-1 mRNA in the lateral wall, organ of Corti and spiral ganglion. This distribution was confirmed by immunocytochemistry. Taken together, these data identify kidney injury molecule-1 as a novel cochlear injury molecule, whose expression is regulated by reactive oxygen species generated via the NADPH oxidase pathway.
Asunto(s)
Antineoplásicos/farmacología , Moléculas de Adhesión Celular/metabolismo , Cisplatino/farmacología , Cóclea/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Animales , Antioxidantes/uso terapéutico , Northern Blotting/métodos , Moléculas de Adhesión Celular/genética , Interacciones Farmacológicas , Pérdida Auditiva/tratamiento farmacológico , Pérdida Auditiva/fisiopatología , Inmunohistoquímica/métodos , Masculino , Proteínas de la Membrana/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ácido Tióctico/uso terapéutico , Factores de TiempoRESUMEN
During postnatal development of rat cochlear cells and the onset of hearing (10-23 days), the increasing endocochlear potential and energy requirements are largely provided by increased glucose utilization. It is well established that the ability of maturing rat tissues to use glucose is directly related to alteration of 6-phosphofructo-1-kinase (PFK) subunits. To gain insight into the alteration of PFK subunit levels in the cochlea from 6 to 60 days of age, PFK subunit types were measured in sections of paraffin-embedded temporal bone using IgG specific for each type of PFK subunit and quantified by computer image analysis. Although the L-type and C-type subunits did not exhibit statistically significant changes in the cochlear structures during maturation, the levels of M-type subunit in the stria vascularis cells, spiral ligament cell types I, II, and III, outer hair cells, inner hair cells, and support cells significantly increased. Also, the type IV and V spiral ligament fibrocytes during this period did not exhibit significant alterations of the M-type subunit. These data suggest that during neonatal development of the cochlear, the elevated levels of the M-type subunit are associated with increased glucose utilization and the onset of hearing.
Asunto(s)
Cóclea/enzimología , Cóclea/crecimiento & desarrollo , Fosfofructoquinasa-1/metabolismo , Animales , Animales Recién Nacidos , Cóclea/citología , Metabolismo Energético , Glucosa/metabolismo , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/crecimiento & desarrollo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/crecimiento & desarrollo , Células Ciliadas Auditivas Externas/metabolismo , Audición/fisiología , Inmunohistoquímica , Fosfofructoquinasa-1/química , Subunidades de Proteína , Ratas , Ratas Endogámicas F344 , Estría Vascular/citología , Estría Vascular/metabolismo , Distribución TisularRESUMEN
During muscle, heart, and brain neonatal maturation, the capacity to utilize glucose in energy metabolism is directly related to the extent of accumulation of the 6-phosphofructo-1-kinase (PFK) M-type subunit. Neonatal development of other organs, such as liver and kidney, which are not characterized by large increases in the capacity to use glucose do not exhibit large increases in the M-type subunit protein. The presence of the M-type subunit in a PFK isozyme pool fosters a higher affinity utilization of carbohydrate and increased responsiveness to the levels of regulatory metabolites. To better appreciate this phenomenon, which is vital for normal development, the different isoforms of the M-type subunit mRNA's and alteration of their levels during maturation have been examined. Further, the potential promoter regions, i.e., the regions upstream from the sites of initiation of transcription, which are involved in expression of the different M-type subunit mRNA isoforms have been isolated, sequenced, and examined for possible transcription factor interaction sites. Using cDNA libraries produced from adult rat brain or skeletal muscle RNA, two primary forms of rat M-type subunit cDNA's were detected. Although the translated regions of these mRNA's were essentially identical, the 5'-untranslated region (5'-UTR) exhibited different lengths (90 or 59 bp) and sequences. Each M-type subunit cDNA had 10 common nucleotides immediately upstream from the initiator ATG, and the remaining 5'-UTR's had insignificant identity. A genomic fragment which interacted with probes complimentary to the sequences of the 5'-UTR of each M-type subunit mRNA isoform was isolated and sequenced by primer walking. It was discovered that the 5'-UTR of one of the mRNA's (proximal mRNA) was located immediately upstream from exon I and was apparently transcribed without splicing. Subsequently, the initial bp in the sequence of the other mRNA isoform (distal mRNA) was located 4010 bp upstream from the ATG in exon 1. Employing Reverse Transcription-Polymerase Chain Reaction using total RNA and scanning densitometry, the relative levels of the proximal and distal mRNA's during neonatal maturation of brain, heart, and muscle were measured. In these tissues, both forms of M-type subunit mRNA's were present, and during maturation tissue-specific differences were noted.
Asunto(s)
Fosfofructoquinasa-1/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Animales , Animales Recién Nacidos , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , ADN Complementario/genética , Corazón/crecimiento & desarrollo , Isoenzimas/química , Isoenzimas/genética , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosfofructoquinasa-1/química , Subunidades de Proteína , Ratas , Ratas Endogámicas F344RESUMEN
For the three 6-phosphofructo-1-kinase (PFK) subunits in heart, skeletal muscle, liver and kidney, developmentally-associated changes in protein, mRNA and apparent synthesis rates were observed. During neonatal maturation, all three phenomena for the M-type in heart and skeletal muscle exhibited large increases. Also, during neonatal development, the L-type and C-type subunits were unaffected in heart but disappeared from skeletal muscle. In the newborn liver and kidney, the amounts of each type of PFK subunit protein were nearly identical. During neonatal development, the levels of all three PFK subunit proteins in kidney increased more than twofold; and this was associated with a similar increase in apparent subunit synthesis rates and mRNA levels. During liver neonatal development, the L-type subunit protein, synthesis and mRNA levels also increased more than twofold. However, during hepatic maturation, M-type subunit protein, synthesis and mRNA levels were unchanged and apparently unaffected. The C-type subunit protein during neonatal liver development decreased approximately 80% as did its apparent synthesis rate. These data suggest that regulation of the alteration of the PFK subunit proteins during neonatal maturation can vary among these tissues and is not the same for each subunit type. Different mechanisms, such as transcription, translation, and mRNA stability could be involved.
Asunto(s)
Músculo Esquelético/metabolismo , Fosfofructoquinasa-1/metabolismo , ARN Mensajero/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos/metabolismo , Hígado/metabolismo , Miocardio/metabolismo , Ratas , Ratas WistarRESUMEN
Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Isoenzimas/metabolismo , Fosfofructoquinasa-1/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Western Blotting , Células Clonales , Activación Enzimática , Fructosadifosfatos/farmacología , Fructosafosfatos/farmacología , Glucosa/farmacología , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Isoenzimas/aislamiento & purificación , Cinética , Sustancias Macromoleculares , Masculino , Oscilometría , Fosfofructoquinasa-1/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
During neonatal maturation of rat brain, a similar biphasic relationship exists between the previously reported pattern of glucose utilization and levels of each type of 6-phosphofructo-1-kinase (PFK) subunit protein, relative synthesis, and mRNA. The increasing amounts of each subunit isoform generally correlated with elevated protein synthesis which was promoted by greater amounts of each type of subunit mRNA. For each parameter, the early phase, 1 to 10 days after birth, was characterized by small increases, and the subsequent period from ten to thirty days postpartum was characterized by a much greater rate of increase. By 30 days after birth, adult values were observed. The apparent efficiency of translation of each type of PFK subunit mRNA in brain suggests that the M-type subunit mRNA is the most efficient and that the L-type subunit mRNA is the least. The greatest relative increases in subunit protein, mRNA, and synthesis were observed for the C-type subunit. Since enhanced translation apparently makes little, if any, contribution, a possible explanation of these phenomena could be increased transcription of the PFK genes. These neonatal changes could involve age-dependent alteration of methylation of the PFK gene promotor(s) and/or activity of effectors of the transcription of the PFK genes.
Asunto(s)
Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Encéfalo/enzimología , Fosfofructoquinasa-1/biosíntesis , Fosfofructoquinasa-1/metabolismo , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Metabolismo Energético/fisiología , Datos de Secuencia Molecular , Fosfofructoquinasa-1/ultraestructura , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
During the 6 days following birth, tissue levels of fructose-2,6-P2 in rat brain, liver, muscle, heart and kidney did not significantly change. However, by the tenth day postpartum fructose-2,6-P2 levels in brain, heart, and skeletal muscle increased approximately 50% and attained adult values. During maturation of liver, adult levels of fructose-2,6-P2 were not achieved until 3-4 weeks after birth or approximately at the time of maximum rates of gluconeogenesis. Renal fructose-2,6-P2 levels in the neonate were initially elevated and 2-3 weeks after birth decreased approximately 2.5-fold to adult values. With the exception of the pons-medulla, which showed no significant changes in fructose-2,6-P2 amounts, levels of this regulatory sugar from aging brain regions were generally decreased. The fructose-2,6-P2 levels from heart atria of old rats (24-30 month) were also significantly decreased. In diaphragm, the fructose-2,6-P2 levels were increased at 12 months of age and at 27 months of age were twice the level at 3 months. The fructose-2,6-P2 levels during the aging of liver, skeletal muscle (EDL and soleus), spleen, thymus, kidney, testis and lung were not significantly altered.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Fructosadifosfatos/metabolismo , Fosfofructoquinasa-1/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Especificidad de Órganos/fisiología , Ratas , Ratas Endogámicas F344 , Ratas WistarRESUMEN
Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P2 in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P2 in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P2 levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P2 due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/enzimología , Fructosadifosfatos/metabolismo , Fosfofructoquinasa-1/metabolismo , Animales , Metabolismo Energético , Cinética , Ratas , Ratas Endogámicas F344RESUMEN
1. The subunit proportions (L:M:C) of the PFK isozymes from normal adult erythrocytes were 2:86:12. Affected adult erythrocyte 6-phosphofructo-1-kinase (PFK) isozymes contained normal L-type (31%) and C-type (61%) subunits as well as a small amount (8%) of truncated M-type subunit. 2. When measured within 24 hr of birth, both normal and affected dog erythrocytes contained high PFK activities due to elevated levels of the L-type subunit. As the dogs matured, PFK activity decreased due to a greater than 99% loss of the L-type subunit. 3. By 2 weeks of age, the M-type and C-type subunits in normal dog PFK isozymes increased several-fold and attained near adult levels. 4. During post-natal development, the L-type subunit from affected dog erythrocytes decreased more rapidly than from normal dog erythrocytes; but it was maintained at a higher level in the affected adult erythrocytes. Also, in the affected dog erythrocytes, truncated M-type subunits were detected; and the initially high levels of the C-type subunit decreased approximately 50% after 4 weeks.
Asunto(s)
Eritrocitos/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo VII/enzimología , Fosfofructoquinasa-1/metabolismo , Envejecimiento/metabolismo , Animales , Modelos Animales de Enfermedad , Perros , Immunoblotting , Valores de ReferenciaRESUMEN
Dogs homozygously affected with muscle-type phosphofructokinase (PFK) deficiency had about 20% of normal erythrocyte PFK activity and exhibited a compensated haemolytic anaemia. Erythrocyte glucose-6-phosphate and fructose-6-phosphate concentrations were increased and dihydroxyacetone phosphate and 2,3-bisphosphoglycerate values were below normal in affected dogs. Other intermediates distal to the PFK step were not significantly below normal and fructose-1,6-bisphosphate was even above normal. Erythrocyte ATP was higher than normal in affected dogs owing to the reticulocytes present. Abnormal adenylate metabolism was demonstrated by low ATP/AMP and ADP/AMP ratios and the inability to maintain ATP content when affected erythrocytes were incubated with cyanide. Glucose-1,6-bisphosphate content was normal, and fructose-2,6-bisphosphate content in affected canine erythrocytes was higher than normal. Studies of erythrocyte PFK isozymes revealed altered enzyme kinetic properties in affected dogs which appeared to be due to the loss of the M-type subunit.
Asunto(s)
Eritrocitos/enzimología , Fosfofructoquinasa-1/deficiencia , Nucleótidos de Adenina/sangre , Amoníaco/sangre , Animales , Recuento de Células Sanguíneas , Portador Sano , Perros , Glucólisis , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Cianuro de Potasio/farmacologíaRESUMEN
Relative to 2-3 month rats, total 6-phosphofructo-1-kinase (PFK) activity in heart atria from 12 month rats declined 31%; but, by 24 months it was decreased by only 13%. PFK activities from 12 and 24 month ventricles relative to the 2-3 month rats were decreased by 40% and 30%, respectively. This change in PFK activity in each heart region was associated with alterations of subunit composition. In heart atria from 12 and 24 month rats when compared to 3 month rats, the levels of L-type subunit were not significantly different; but the levels of the M-type subunit were decreased by 43% and 38%, respectively. With respect to levels in 2-3 month atria, the C-type subunit in 12 month atria decreased by 27%; and at 24 months it increased by 31%. Making the same comparison for the heart ventricle at 12 and 24 months, L-type subunit decreased by 30% and 24% respectively; M-type subunit decreased by approximately 47%; and the C-type subunit increased 1.9 and 4.7 fold, respectively. These age-related changes of subunit composition in atrial and ventricular PFK isozyme pools led to changes in their kinetic and regulatory properties suggesting that the aged rat could exhibit a diminished capacity to produce ATP from glucose.
Asunto(s)
Envejecimiento/metabolismo , Atrios Cardíacos/enzimología , Ventrículos Cardíacos/enzimología , Isoenzimas/aislamiento & purificación , Fosfofructoquinasa-1/aislamiento & purificación , Animales , Encéfalo/enzimología , Isoenzimas/metabolismo , Cinética , Hígado/enzimología , Especificidad de Órganos , Fosfofructoquinasa-1/metabolismo , Ratas , Ratas Endogámicas/metabolismoRESUMEN
6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31% of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.
Asunto(s)
Encéfalo/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo VII/enzimología , Fosfofructoquinasa-1/química , Adenosina Trifosfato/farmacología , Animales , Perros , Fructosadifosfatos/farmacología , Fructosafosfatos/farmacología , Isoenzimas , Cinética , Fosfofructoquinasa-1/efectos de los fármacos , Fosfofructoquinasa-1/genéticaRESUMEN
We report that the short-term use of various anesthetic agents prior to decapitation causes alteration of the levels of fructose-2,6-bisphosphate in kidney, brain, heart, muscle, and liver. These data indicate that even light anesthesia can not be used when levels of this metabolite are to be determined. Also, it appears that the use of any of these anesthetics can profoundly alter glucose utilization in many tissues.
Asunto(s)
Anestésicos/farmacología , Fructosadifosfatos/análisis , Hexosadifosfatos/análisis , Ratas Endogámicas , Animales , Química Encefálica , Hidrato de Cloral/farmacología , Cloralosa/farmacología , Eutanasia/veterinaria , Halotano/farmacología , Ketamina/farmacología , Riñón/análisis , Hígado/análisis , Músculos/análisis , Miocardio/análisis , Pentobarbital/farmacología , RatasRESUMEN
6-Phosphofructo-1-kinase (PFK) isoenzyme pools from livers of fetal, neonatal, young adult (3 months) and aged (24 months) rats were studied. Near-term liver PFK isoenzyme pools were composed of nearly equal quantities of all three subunits. During the 30 days after birth, the total activity increased by 25%; the amount of the L-type, M-type or C-type subunit was increased 3-fold, was unchanged, or was decreased by 80% respectively. In aged rats, compared with young adults, total PFK activity was unchanged, but the L-type, M-type or C-type subunit decreased by 24%, increased by 39%, or increased by 338% respectively. During neonatal maturation, the changing subunit composition of the hepatic isoenzyme pools led to a decreased susceptibility to ATP inhibition, to a greater apparent affinity for fructose 6-phosphate, and to increased sensitivity to fructose 2,6-bisphosphate. Also, these alterations correlated with the measured increases in fructose 2,6-bisphosphate and the reported optimal rate of hepatic glycolysis/gluconeogenesis.
Asunto(s)
Envejecimiento , Isoenzimas/metabolismo , Hígado/enzimología , Fosfofructoquinasa-1/metabolismo , Animales , Femenino , Feto , Fructosadifosfatos/metabolismo , Glucólisis , Embarazo , Ratas , Ratas EndogámicasRESUMEN
During postnatal development, the subunit compositions of the 6-phosphofructo-1-kinase isozyme pools of heart and skeletal muscle are known to change. The isozyme pools from fetal muscle were composed of the L-type (60%), and M-type (36%) and C-type (4%) subunits and the isozymes from fetal and early neonatal heart contain nearly equal amounts of all three subunits. During postnatal development of both tissues, the proportion of the M-type subunit increases until it is the only type present in adult muscle and the major subunit in adult heart (75%). The isozyme pool from fetal muscle exhibit a decreased affinity for fructose-6-P and a greater susceptibility to ATP inhibition compared to the M-rich isozymes which are subsequently present. The isozyme pools from fetal and early neonatal heart, if compared to the M-rich isozymes which are present later during heart development and to the fetal muscle isozymes, exhibited the least affinity for fructose-6-P and the greatest susceptibility to ATP inhibition. Comparison of the isozyme pools containing little or no C-type subunit with those from fetal and early neonatal heart clearly indicates that the presence of substantial levels of the C-type subunit imposed a decreased ability for fructose-2,6-P2 to both lower affinity for fructose-6-P and antagonize sensitivity to ATP inhibition. Although still not thoroughly appreciated, it appears that the changing nature of the isozyme pools in these tissues permits regulation of glucose metabolism in a manner which allows efficient utilization of nutritional opportunities and which adequately meets the energy requirements of each tissue at different stages of development.
Asunto(s)
Corazón/embriología , Isoenzimas/metabolismo , Músculos/embriología , Miocardio/enzimología , Fosfotransferasas/metabolismo , Adenosina Trifosfato/fisiología , Animales , Femenino , Cinética , Músculos/enzimología , Embarazo , Conformación Proteica , Ratas , Ratas EndogámicasRESUMEN
The 6-phosphofructo-1-kinase (PFK) isozyme pools from brains of fetal, neonatal, young adult (3 months) and aged (30 months) rats were studied using chromatographic and immunological techniques. Also, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. The total PFK activity increased over seven-fold during the 30 days following birth, and the L-type, M-type, and C-type subunits increased approximately 2-fold, 7-fold, and 24-fold, respectively. In the near-term fetal brain and early neonatal brain, the L-type and M-type subunits were the predominant forms and were present in approximately equal amounts. During the second second week of postnatal brain maturation, the levels of the M-type and C-type subunit began to significantly increase. Consequently, during postnatal development, the isozyme pools switched from L-M-rich forms to M-C-rich forms. In aged brain relative to the young adult (3 months) brain, the 20% loss of total activity was associated with 27% and 18% losses of the M-type and C-type subunits, respectively. Examination of the regulatory properties of the various PFK isozyme pools revealed that at the low concentration of fructose-6-P and high level of ATP which are thought to occur in vivo, fructose-2,6-P2 was required for measurable PFK activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Encéfalo/crecimiento & desarrollo , Isoenzimas/metabolismo , Fosfofructoquinasa-1/metabolismo , Envejecimiento , Animales , Encéfalo/embriología , Encéfalo/enzimología , Cinética , Sustancias Macromoleculares , Ratas , Ratas Endogámicas , Valores de ReferenciaRESUMEN
The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.
Asunto(s)
Isoenzimas/metabolismo , Fosfofructoquinasa-1/metabolismo , Adenosina Trifosfato/farmacología , Adolescente , Electroforesis en Gel de Poliacrilamida , Fructosafosfatos/metabolismo , Humanos , Inmunoglobulina G , Isoenzimas/antagonistas & inhibidores , Isoenzimas/inmunología , Masculino , Fosfofructoquinasa-1/antagonistas & inhibidores , Fosfofructoquinasa-1/inmunología , Distribución TisularRESUMEN
The nature of the PFK (6-phosphofructo-1-kinase) isoenzymes in many rat tissues was examined by immunological and chromatographic techniques and by measurement of their subunit compositions. It was revealed that, except for diaphragm and skeletal muscle, these complex isoenzymic populations contained different amounts of the three subunit types and were nearly tissue-specific. Apparently this tissue specificity is due to different concentrations of the tetramers, which in turn are controlled by the types and amounts of each subunit that are available to associate randomly.
Asunto(s)
Isoenzimas , Fosfofructoquinasa-1 , Animales , Electroforesis en Gel de Poliacrilamida , Inmunoelectroforesis , Especificidad de Órganos , Proteínas/análisis , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
The nature of 6-phosphofructo-1-kinase isozyme pools in fetal, neonatal, young adult (3 months), and aged (30 months) rat hearts was studied using chromatographic and immunological techniques. Furthermore, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. Although all three subunit types were expressed in heart throughout life, total activity and the nature of the isozyme pools varied during neonatal development and in aged heart. In fetal heart, the complex tetramers containing all three subunits appeared to be the major isozyme types. As the heart matured to the young adult stage, the M-type subunit increased over 6-fold; whereas the changes in the other two subunits were considerably less. These data indicate that during neonatal heart maturation the isozymic pools progressively exhibited increased amounts of the tetrameric forms containing two or more M-type subunits. In aged heart relative to the young adult (3 months) heart, the total activity and proportion of M-type subunit in the isozymes were decreased; and consequently, the amounts of the M-rich isozymes were decreased. The shifts in the types of isozymes during heart maturation and subsequent aging were primarily due to changes in availability of the M-type subunit to participate in random assembly of the tetrameric isozymes.
Asunto(s)
Corazón/crecimiento & desarrollo , Isoenzimas/metabolismo , Miocardio/enzimología , Fosfofructoquinasa-1/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Feto , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas , Especificidad de la EspecieRESUMEN
The purpose of this paper is to provide insight into the alterations of 6-phosphofructo-1-kinase (PFK) activity and isozyme types of rat skeletal muscle during development and aging. PFK isozymes are tetramers which may be comprised of one or any combination of the three subunit types, L, M, and C. The effects of fusion or terminal differentiation of cultured rat L6 myoblasts leading to formation of myotubles does not have a noticeable effect on total PFK activity. However, the amount of M-type subunit was increased; and the level of the C-type subunit decreased. These subunit changes caused shifts in the isozymic types. The ultimate effects of prenatal development of PFK were characterized in the near-term fetal muscle. This stage of development was accompanied by a significant loss of the C-type subunit and by two-fold increases in the L-type and M-type subunits which accounted for the 40% increase in total PFK activity. After birth, the M-type subunit increased dramatically as did the total PFK activity. Since the L-type and C-type subunits were gradually lost during the subsequent 3 weeks, the homotetramer of the M-type subunit (M4) was the only type which is present in mature muscle. M4 persisted as the only detectable form in skeletal muscle during the remainder of life, but the total PFK activity and amount of M4 decreased after 18 months of age. The decreased total PFK activity in aged skeletal muscle suggested that the expression of PFK genes may have reverted to an immature state when total PFK activity was lower. As shown by both the immunological analysis and direct quantification of subunit types, this clearly did not occur. That is, the loss of PFK activity in aged muscle is a consequence of decreased levels of the M-type subunit and not reappearance of other subunit types such as found in maturing muscle. Further, our examination of aged skeletal muscle indicates that no significant structural changes in M-type subunits had occurred and that inactive or partially active proteins which could crossreact with the M-type subunit were not detectable. It is suggested that the loss of M4 could cause a depression of the glycolytic rate leading to diminished ability of senile muscle to accommodate extreme energy demands.