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1.
Cytochrome c oxidase was isolated from brown fat tissue of the rat and compared with the isozymes from rat liver and heart, which differ at least in subunits VIa and VIII. ELISA titrations of COX from the three tissues with monospecific antisera to all 13 subunits of the rat liver enzyme showed differences between the three enzymes. The N-terminal amino-acid sequence analysis of subunits VIa and VIII from SDS-PAGE gel bands of the three enzymes indicates the occurrence of three different isozymes in the rat. N-terminal amino-acid sequence analysis of subunits VIa and VIII from cytochrome c oxidase of bovine and human heart demonstrates also species-specific differences in the expression of the 'liver-type' and 'heart-type' of subunits VIa and VIII.  相似文献   

2.
A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate that heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.  相似文献   

3.
Human 6-phosphofructokinase (EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M), liver (L) and platelet (P) subunits, which are under separate genetic control. In the adult, the proportion of these subunits in different organs reflects the relative activity of glycolysis versus gluconeogenesis. To elucidate the developmental basis for the observed distribution, we investigated the isoenzymic transitions of phosphofructokinase in human foetuses (12-40 weeks' gestation) by using high-resolution chromatography and monoclonal antibodies. We studied skeletal muscle, heart, liver and brain because these organs show very different glycolytic fluxes and isoenzymic patterns in adult individuals. Our results demonstrate that there is no unique 'foetal' form of phosphofructokinase in humans, but all three loci are variably expressed in all foetal organs during early gestation. As development proceeds, muscle and liver isoenzyme patterns show dramatic changes, with disappearance of P and L subunits in muscle and transient reappearance of M and P subunits in liver; in contrast, phosphofructokinase isoenzymes change little in brain and heart. Most changes occur at mid-gestation and near term, and adult isoenzyme patterns are expressed at birth, indicating that organ differentiation is complete. These studies show that phosphofructokinase undergoes changes of isoenzyme patterns similar to, but not identical with, those of other multilocus isoenzyme systems of glycolysis. The observed changes probably reflect changing patterns of gene expression, with repression of some loci and activation of others.  相似文献   

4.
L (liver) type phosphofructokinase subunits purified from human leukocytes are slightly lighter than L subunits from liver and red blood cells. A mild treatment of red blood cell L4 enzyme with subtilisin converts its subunits into forms of similar molecular weight to leukocyte enzyme. From a kinetical point of view, subtilisin-treated L4 phosphofructokinase and leukocyte enzymes are characterized by a decrease of the allosteric properties as compared to non-treated red cell L4 phosphofructokinase.  相似文献   

5.
A comparative analysis of distribution of lactate dehydrogenase (LDH) isoenzymes in the heart, kidneys, lungs, spleen, liver and skeletal muscle was performed in 4 species of carnivorous mammals (Mustelidae family): American mink (Mustela vison Schr.), polecat (Mustela putorius L.), sable (Martes zibellina L.) and pine marten (Martes martes L.). It was found that in the mink, unlike pure terrestrial mustelids (polecat, sable, pine marten), the anaerobic LDH-5 fraction was predominant in the LDH isoenzyme spectrum in the liver, skeletal muscle, lungs and spleen. In all species studied, aerobic B(H) subunits of LDH were found to prevail in the heart and kidney, whereas anaerobic A(M) subunits prevailed in the liver and spleen. The tissue- and species-specific features of LDH isoenzyme spectra revealed in different mustelids reflect the biochemical adaptation of the animals to environmental conditions.  相似文献   

6.
Tissue-specific differences between heart and liver cytochrome c oxidase   总被引:5,自引:0,他引:5  
Bovine liver cytochrome c oxidase has been isolated and the subunit structure of this preparation compared with that of the bovine heart enzyme. Of the 10 nuclear-coded subunits, 3 were different in the 2 tissue forms, having different migrations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, different antigenicities to antibodies made against the heart subunits, and different N-terminal amino acid sequences. Subunit ASA of heart begins with the N-terminal sequence of SSG in liver and is different in 17 of the first 33 residues including a deletion of 2 residues in the liver isoform of this subunit. Subunit CVII of liver differs from its heart counterpart in 6 of the first 37 residues while subunit CIX from liver differs from the heart isoform in 15 of the first 25 residues. No differences between tissue types were observed in partial sequencing of the remaining nuclear-coded subunits. Recently, the major portion of the sequence of subunit CIX from rat liver has been obtained by cloning and sequencing of the cDNA for this polypeptide [Suske, G., Mengel, T., Cordingley, M., & Kadenbach, B. (1987) Eur. J. Biochem. 168, 233-237]. There is a greater sequence homology of the rat and bovine liver forms of CIX than there is between the bovine heart and liver isoforms.  相似文献   

7.
Summary Protein phosphatase 2A1 was purified from rat skeletal muscle and used to produce antisera to the three subunits of the holoenzyme. Affinity purified antibodies specific for the subunits of the phosphatase enzyme were found to recognize the type 2A1 and 2A2 phosphatase from rat skeletal muscle, heart, liver, brain and erythrocytes and were used to investigate the effects of diabetes on the levels of this enzyme in liver and heart. Phosphorylase phosphatase assays coupled with immunoblot analysis of fractionated rat liver and heart cytosol from normal and diabetic animals show no apparent differences in the quantity or activity of these enzymes following the induction of alloxan diabetes. When considering these results and the normal physiological concentrations of known effectors of these enzymes, it is likely that protein phosphatase 2A1 and 2A2 are not responsible for the dephosphorylation of phosphorylase a under physiological conditions.  相似文献   

8.
In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.  相似文献   

9.
Extremities, peptide maps and phosphorylatable site localization of human erythrocyte L' and liver L pyruvate kinases (EC 2.7.1.40) were investigated. L' and L subunits seemed to have similar, blocked NH2 termini and differ in their sensitivity to carboxypeptidase A, that is to say in their C-terminal ends. After digestion by Staphylococcus aureus V8 protease, the phosphorylated sites of both L' and L subunits were located on those peptides which were different in L' and L, that is to say on the C-terminal sides. A mild proteolytic attack of the native tetrameric enzymes by trypsin partially degraded the phosphorylatable peptides without removing the phosphoserine residue; in the same conditions, chymotrypsin split off this phosphorylated residue and subtilisin totally degraded the phosphorylated peptides. From these results it appears, therefore, that age-dependent proteolytic degradation of L' subunits in old red cells involves the C-terminal side of the molecules, ultimately resulting in cleavage of the phosphorylated site. Since erythrocyte L' and liver L subunits are encoded by different species of messenger RNAs, our results indicate, in addition, that these messenger RNA species should differ by their 3' coding sequences.  相似文献   

10.
The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.  相似文献   

11.
A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.  相似文献   

12.
(1) The tissue-specific expression of various glutathione-dependent enzymes, including glutathione S-transferase (GST), glutathione peroxidase and glyoxalase I, has been studied in bovine adrenals, brain, heart, kidney, liver, lung and spleen. Of the organs studied, liver was found to possess the greatest GST and glyoxalase I activity, and spleen the greatest glutathione peroxidase activity. The adrenals contained large amounts of these glutathione-dependent enzymes, but significant differences were observed between the cortex and medulla. (2) GST and glyoxalase I activity were isolated by S-hexylglutathione affinity chromatography. Glyoxalase I was found in all the organs examined, but GST exhibited marked tissue-specific expression. (3) The alpha, mu and pi classes of GST (i.e., those that comprise respectively Ya/Yc, Yb/Yn and Yf subunits) were all identified in bovine tissues. However, the Ya and Yc subunits of the alpha class GST were not co-ordinately regulated nor were the Yb and Yn subunits of the mu class GST. (4) Bovine Ya subunits (25.5-25.7 kDa) were detected in the adrenal, liver and kidney, but not in brain, heart, lung or spleen. The Yc subunit (26.4 kDa) was expressed in all those organs which expressed the Ya subunit, but was also found in lung. The mu class Yb (27.0 kDa) and Yn (26.1 kDa) subunits were present in all organs; however, brain, lung and spleen contained significantly more Yn than Yb type subunits. The pi class Yf subunit (24.8 kDa) was detected in large amounts in the adrenals, brain, heart, lung and spleen, but not in kidney or liver. (5) Gradient affinity elution of S-hexylglutathione-Sepharose showed that the bovine proteins that bind to this matrix elute in the order Ya/Yc, Yf, Yb/Yn and glyoxalase I. (6) In conclusion, the present investigation has shown that bovine GST are much more complex than previously supposed; Asaoka (J. Biochem. 95 (1984) 685-696) reported the purification of mu class GST but neither alpha nor pi class GST were isolated.  相似文献   

13.
A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.  相似文献   

14.
Abstract Heterologous DNA hybridization using a ribulose- 1,5-biphosphate carboxylase/oxygenase (RuBisCOase) large subunit gene ( rbc L) probe from Anacystis nidulans revealed the presence of two rbc L in Nitrobacter hamburgensis . One gene is located on a plasmid, the other on the chromosome. The genes appear to be very similar since both hybridized strongly to the A. nidulans probe. However, restriction endonuclease digestions revealed differences.
Two different RuBisCOase enzymes were isolated from N. hamburgensis. The M r of the native enzymes were 520 000 and 480 000. Sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) revealed the presence of both LSU and small subunits (SSU) for both enzymes. The M r were 53 000 and 16 000, and 49 000 and 13 500, respectively. A hexadecameric structure is suggested for both enzymes.  相似文献   

15.
The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I–III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mito-chondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g., VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochromec oxidation by the reconstituted enzyme from bovine heart but not fromP. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochromec oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochromec oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.  相似文献   

16.
Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.  相似文献   

17.
Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. Expression of one family member, PTG, in the liver of normal rats improves glucose tolerance without affecting other plasma variables but leaves animals unable to reduce hepatic glycogen stores in response to fasting. In the current study, we have tested whether expression of other targeting subunit isoforms, such as the liver isoform G(L), the muscle isoform G(M)/R(Gl), or a truncated version of G(M)/R(Gl) termed G(M)DeltaC in liver ameliorates glucose intolerance in rats fed on a high fat diet (HF). HF animals overexpressing G(M)DeltaC, but not G(L) or G(M)/R(Gl), exhibited a decline in blood glucose of 35-44 mg/dl relative to control HF animals during an oral glucose tolerance test (OGTT) such that levels were indistinguishable from those of normal rats fed on standard chow at all but one time point. Hepatic glycogen levels were 2.1-2.4-fold greater in G(L)- and G(M)DeltaC-overexpressing HF rats compared with control HF animals following OGTT. In a second set of studies on fed and 20-h fasted HF animals, G(M)DeltaC-overexpressing rats lowered their liver glycogen levels by 57% (from 402 +/- 54 to 173 +/- 27 microg of glycogen/mg of protein) in the fasted versus fed states compared with only 44% in G(L)-overexpressing animals (from 740 +/- 35 to 413 +/- 141 microg of glycogen/mg of protein). Since the OGTT studies were performed on 20-h fasted rats, this meant that G(M)DeltaC-overexpressing rats synthesized much more glycogen than G(L)-overexpressing HF rats during the OGTT (419 versus 117 microg of glycogen/mg of protein, respectively), helping to explain why G(M)DeltaC preferentially enhanced glucose clearance. We conclude that G(M)DeltaC has a unique combination of glycogenic potency and responsiveness to glycogenolytic signals that allows it to be used to lower blood glucose levels in diabetes.  相似文献   

18.
The objective of this study was to investigate the in vitro effects of the organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one on some parameters of oxidative stress in liver, kidney, and heart of 10-day-old rats. The homogenates of liver, kidney, and heart were incubated for 1 h in the absence (control) or in the presence of 1, 10, or 30 μM of the organoselenium and thiobarbituric acid reactive substances, carbonyl, and the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured. First, we tested the influence of the compound on 1,1-diphenyl-2-picrylhydrazyl (DPPH(?)) radical scavenging and verified that the organochalcogen did not have any antioxidant properties. We observed an increase of lipid peroxidation in all concentrations tested in heart and kidney, while in liver only in the concentrations of 10 and 30 μM. Moreover, we also verified an enhance of protein oxidation in the concentrations of 10 and 30 μM in kidney. On the other hand, the compound caused a reduction on the activity of CAT in heart (10 and 30 μM), liver (30 μM), and kidney (30 μM). The activity of SOD was increased in heart (10 and 30 μM), while in liver (30 μM) and in kidney (10 and 30 μM) the activity was reduced. Our findings indicate that this organoselenium compound induces oxidative stress in liver, heart, and kidney of immature rats, collaborating to the fact that these tissues are potential targets for the organochalcogen action.  相似文献   

19.
Cytochrome c oxidase was isolated from pig, bovine, rat and human tissues including liver, heart, diaphragm and kidney. The native and the sodium-dodecyl-sulfate (SDS)-dissociated enzymes were labelled under optimal conditions with N-ethyl-[2,3-14C]maleimide before and after reduction with dithiothreitol, separated into 13 subunits by SDS gel electrophoresis and the radioactive bands were visualized by fluorography. In some cases the radioactive bands were cut out and counted. All isozymes were labelled in subunits I, III, Va and VIIb, and in subunit II after reduction. Labelling of subunit Vb was equivocal, and in no case were subunits IV and VIc labelled. All other subunits were labelled tissue-specifically and/or species-specifically. No differences were found between labelling of the native and SDS-dissociated enzyme. By relating the molar amount of bound N-ethylmaleimide to the known amount of cysteines in subunits of bovine heart cytochrome c oxidase, the percentage of -SH group reactivity was calculated. Only the cysteine of subunit Va was found to be 100% reactive. The distinct and different reactivity of subunit VIIb as compared to subunits VIIa and VIIc clearly establishes this polypeptide as an independent subunit of mammalian cytochrome c oxidase.  相似文献   

20.
本文以聚丙烯酰胺凝胶电泳法分析华南兔(Lepus sinensis sinensis)11种组织乳酸脱氢酶同工酶的分布待征。分析结果:骨骼肌和肝组织5条同工酶带俱全;脑、肺、卵巢和盲肠等组织各含有4条谱带(LDH-1,-2,-3和-4);胃和肾组织含有3条谱带(LDH-1,-2,和-3);眼晶状体和睾丸组织也含有3条谱带,但前者是LDH-3,-4和-5,后者是LDH-1,-2和-5;谱带最少的是心肌组织,只有2条(LDH-1和-2)。此外,还对各组织中的亚基活性分布及电泳图谱特征进行了分析。  相似文献   

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