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1.
The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.  相似文献   

2.
The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.  相似文献   

3.
Recent studies suggest that the tissue/organ proportions of 6-phosphofructo-1-kinase (PFK) subunits from diverse strains of rat may be drastically different. To test this possibility rigorously, the PFK isoenzyme populations and subunit contents in muscle, liver, brain and heart were examined in the following strains: Wistar, ACI, Long Evans, Norway Brown and Wag/Rij. Regardless of the strain, adult muscle possessed only the M-type subunit; adult liver contained predominantly the L-type subunit as well as M-type and C-type subunits; and the adult brain and heart exhibited all three subunit types.  相似文献   

4.
1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific.  相似文献   

5.
本实验从成年小鼠和胎龄4-5月的人胎儿不同器官中分离总RNA。经斑点印迹分析显示,肝细胞生长因子(HGF)mRNA在成年KM小鼠多种器官中表达,其表达水平由高到低依次为:肺、肝、肾、卵巢、睾丸、大脑和胃;在脾、心、骨髓、小肠和骨骼肌组织中以HGFmRNA。在胎龄4-5月的人胎儿中,HGFmRNA表达水平由高到低依次为:大脑、肝、腮腺、胃、小肠、肾、心和骨骼肌;肺和脾组织为阴性。由此可见,HGF在成  相似文献   

6.
As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.  相似文献   

7.
Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).  相似文献   

8.
S Vora  R Oskam    G E Staal 《The Biochemical journal》1985,229(2):333-341
In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.  相似文献   

9.
Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.  相似文献   

10.
Multiple forms of phosphofructokinase in striated muscle and cardiac muscle of developing rabbit (Oryctolagus cuniculus) undergo changes with development, but not in brain and liver. The cardiac muscle of the 1-day-old rabbit contains phosphofructokinase A4 together with the four hybrid forms which were tentatively called A3C, A2C2, AC3, and C4. In older animals, phosphofructokinase C4 disappears first, followed by the hybrid forms, and only phosphofructokinase A4 persists in the adult animal. Both phosphofructokinase A4 and phosphofructokinase C4, as well as their hybrid forms, are present in developing embryonic brain and also in the brains of adult animals. Developing rabbit liver contains a single form of phosphofructokinase, but two isoenzymes are consistently seen in guinea pig liver. In striated muscle from fetal and 1-day-old rabbit, two isoenzymes are found, tentatively identified as A4 and the A3C hybrid. The results suggest that fetal phosphofructokinase A4 and phosphofructokinase C4, and their hybrids, might be present in striated muscle. Guinea pig tissues show a pattern of phosphofructokinase isoenzymes different from that in rabbit tissues.  相似文献   

11.
Developmental changes in heart and muscle phosphofructokinase isozymes   总被引:2,自引:0,他引:2  
Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel electrophoresis, and immunodiffusion with specific antisera. The results of these studies indicate that in skeletal muscle and heart the levels of the major liver phosphofructokinase isozyme (PFK-L2) and the muscle phosphofructokinase isozyme (PFK-M) are dependent on the developmental status of the rat. For example, PFK-L2 and PFK-M are present in fetal and early neonatal skeletal muscle; whereas in adult skeletal muscle, only PFK-M is detectable. By DEAE- cellulose chromatography, PFK-L2 activity was estimated to be 2.4 units/g (41% of total phosphofructokinase activity) in fetal muscle, very low and not resolved from PFK-M in 7-day neonatal muscle, and not detectable in adult muscle. Further, PFK-M activity was found to be 3.4 units/g (59% of total phosphofructokinase activity), 10 units/g, and 31.6 units/g in fetal, 7-day neonatal, and adult skeletal muscle, respectively. The developmental changes of heart phosphofructokinase isozymes differ considerably from that of the skeletal muscle phosphofructokinase isozymes. In fetal heart, PFK-L2 is the major phosphofructokinase isozyme (5.6 units/g), constituting 67% of total phosphofructokinase activity. Further, in fetal heart another phosphofructokinase isozyme (33% of total phosphofructokinase activity) was found by DEAE-cellulose chromatography which is different from PFK-M and PFK-L2. In 7-day neonatal and adult heart, PFK-M and PFK-L2 are the only detectable phosphofructokinase isozymes. Varying from 5.6 units/g (44% of total) in 7-day neonatal to 5.9 units/g (40% of total) in adult heart, PFK-L2 activity remains fairly constant. Also, PFK-M is very low in fetal heart but increases within 1 week postpartum to 5.5 units/g (50% of total activity) and to 8.9 units/g (60% of total activity) in adult heart.  相似文献   

12.
Isoenzyme patterns, total lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity and H and M subunit activity were determined in the tissues of Czech Spotted bovine foetuses. Total LDH activity rose in the skeletal muscles throughout the whole of the prenatal period. In the viscera it usually attained the maximum at a foetal length of 66.7 cm. Differences in the isoenzyme patterns of the various organs of an 8.1-cm foetus were relatively small (41.9--66.1% H subunits). In the heart and kidneys, in which LDH1 and LDH2 markedly predominate in adulthood, the isoenzyme pattern resembled the adult one at a length of only 13.3 cm, but in the liver, spleen and lungs not until 66.7 cm. The proportion of H subunits also rose in the part of the gastrointestinal tract where secretory and resorptive activity predominate (the abomasum, the small and the large intestine). Conversely, it fell in organs concerned mainly with the mechanical processing of food (the rumen, reticulum and omasum). The proportion of M subunits rose in all the skeletal muscles up to a foetal length of 66.7 cm. Later on, differentiation into muscles in which M subunits predominated (the longissimus dorsi and the triceps brachii), into muscles with approximately the same proportion of H and M subunits (the iliopsoas) and to muscles with a preponderance of H subunits (the masseter and the muscular part of the diaphragm) occurred.  相似文献   

13.
1. Six monoclonal antibodies specific to the pyruvate kinase from the foot muscle of the common limpet P. caerulea were produced. 2. They also exhibited specificity against the mouse liver where the L-type isoenzyme of pyruvate kinase is present. They did not react with the mouse skeletal muscle, heart or red blood cells isoenzymes of pyruvate kinase (PK). One of these, the monoclonal antibody B did not react with any PK isoenzymes of the mouse tissues. 3. The presence of the isoenzymic type of PK which was recognized by the monoclonals, (type L), was traced in five phyla of marine invertebrates by the application of the monoclonal antibodies A, B and C. 4. In two phyla the majority of the animals were found to possess an L-type PK isoenzyme in their muscles while in quite a few of them a different isoenzymic type was present in the other tissues. The results of this study are compared with the existing literature, and the use of monoclonal antibodies in the study of enzymic systems is considered in the discussion.  相似文献   

14.
Abstract— Lactate dehydrogenase and malate dehydrogenase isoenzyme patterns of chicken brain and retina have been investigated by cellulose acetate electrophoresis, during embryonic and post-hatching development. The proportion of M-type lactate dehydrogenase subunits decreases significantly in brain and retina with development. A marked increase in the H-type subunits is observed in retina. Lactate dehydrogenase isoenzyme distribution appears to change in both organs in parallel with the metabolic changes of differentiation.
Malate dehydrogenase isoenzyme patterns do not reveal any consistent change of the ratio between the mitochondrial and cytoplasmic forms.  相似文献   

15.
1. Hybridization studies have been carried out in vitro using mixtures of partially purified isoenzymes 1 and 3 of rat enolase (2-phospho-D-glycerate hydrolyase, EC 4.1.1.11). Immunological methods were used to demonstrate the formation of the hybrid. 2. Immunological analysis of the elution peaks from QAE-Sephadex chromatography of heart enolase indicates the occurrence in vivo of the hybrid enolase 2. 3. Developmental changes in the proportions of isoenzymes 1, 2 and 3 in heart and skeletal muscle of rat have been studied quantitatively. In both tissues isoenzyme 1 predominates in the foetus, but is partially replaced by 2 and 3 in adult heart and completely by 3 in the adult muscle. 4. Evidence is given of the binomial distribution of the proportions of the three isoenzymes in the developing heart. 5. Phylogenetic studies of the immunological properties of enolases from muscle, liver and heart have been carried out. 6. It is concluded that the three isoenzymes arise from two independent genetic loci and it is suggested that these evolved from a common ancestral gene 200-300 million years ago.  相似文献   

16.
Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.  相似文献   

17.
18.
The expression of albumin binding proteins (ABP, 31 and 18 kDa peptides) in various organs as a function of their ontogenic development was investigated in fetuses (20 days old), neonates (1 day old) and adult rabbits. At each of these stages, tissue extracts of brain, lung, thymus, heart, skeletal muscle and liver as well as whole embryos (11 days old) were examined by ligand blotting and quantitative immunoblot assays. Blots were either incubated with [125I]albumin followed by autoradiography and radioassay or exposed to a radioiodinated antibody raised against affinity-isolated 31 kDa peptide. Anti-31 kDa IgG cross-reacted with both 31 and 18 kDa peptides. Both methods used revealed that ABP are well expressed in embryos and in all fetal organs investigated. By comparison, in neonates, the ABP expression was diminished (by approximately 2-fold) in brain, heart and skeletal muscle. These changes were even more pronounced in the adult rabbit brain, heart, skeletal muscle and liver; no significant modification was detected in the lung. Prompted by these results, which inferred a high level of ABP in actively proliferating/differentiating tissues, we checked for the presence of ABP in other adult cells and tissues. In bone marrow cells, thymocytes and splenocytes, the 31 and 18 kDa peptides represented the major sodium dodecyl sulfate-urea extracted proteins, whereas in mature circulating white blood cells they were moderately expressed. The results indicate that ABP 1) are present early in embryogenesis, 2) are particularly well expressed in organs (fetal or adult) and cells characterized by active proliferation and differentiation, and 3) are not tissue specific.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
Activity patterns of cytosolic and mitochondrial enzymes of carbohydrate and amino acid metabolism have been measured in murine trisomy 19. In spite of marked hypoplasia, no significant alterations of the patterns (per gram of organ weight) were observed, with the exception of glutamate oxaloacetate transaminase (GOT-1), and phosphoglycerate mutase (PGAM). Clear-cut gene dosage effects in liver, brain, heart, skeletal muscle, and erythrocytes of fetal and newborn mice, confirm the assignment of GOT-1 to chromosome 19. Data obtained for PGAM demonstrate that one of the two different subunits leading to organ-specific isozyme patterns of the dimer enzyme protein is coded on chromosome 19 (gene Pgam-1). Dosage effects are fully expressed in liver, brain, and erythrocytes (AA-type isozyme), but not in skeletal muscle (BB-type isozyme). Dosage effects on the hybrid AA-AB-BB-isozyme pattern in the course of development of the heart muscle, were demonstrated by means of quantitative activity measurement after electrophoretic separation. The comparison of enzyme patterns of eusomic and trisomic erythrocytes, produced after injection of fetal stem cells into irradiated adult carriers (transplantation chimaeras), revealed enzyme activity ratios that were similar to those produced by erythrocytes of adult euploid and trisomic mice. This is in agreement with the chromosome assignments and dosage effects mentioned above.  相似文献   

20.
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