AMP deaminase isozymes in human tissues

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.     

Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

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.     

Demonstration of human alpha-L-fucosidase polymorphism by means of monoclonal antibodies

Conventional rabbit antibodies and mouse monoclonal antibodies were raised to alpha-L-fucosidase purified from human placenta. Four monoclonal antibodies were studied, of which only one (A) was able to immunoprecipitate the fucosidase activity completely. Two antibodies (B and C) precipitated 65% and one (D) 35% of the activity. The enzyme precipitated by the monoclonal antibodies remained fully active, whereas the enzyme precipitated by conventional antibodies was partly inactivated. As shown by the method of successive immunoprecipitations, the monoclonal antibodies B and C recognized the same set of placental fucosidase molecules, and D a subset thereof. The purified fucosidase also yielded two components after gel electrophoresis in nondenaturing conditions, and the slower component corresponded to the set recognized by antibodies B and C. The fucosidase extracted from different tissues and serum was studied by immunoprecipitation. In all cases, the enzyme was completely precipitated by monoclonal antibody A. Two patterns were found with B, C and D: either part of the activity was precipitated by these antibodies (leucocytes, placenta, brain, liver, spleen, thymus) or B, C and D failed to precipitate any of the enzyme (serum, heart, kidney, testes).     

Organ-specific distribution of isozymes of 5'-nucleotide phosphodiesterase in mouse

1. The distribution of isozymes of 5'-nucleotide phosphodiesterase (E.C.3.1.4.1) was examined in various organs of mouse, including liver, spleen, pancreas, heart, lung, kidney, brain and blood. 2. Five isozymes were identified and designated as isozymes I through V. 3. These isozymes are distributed unevenly with respect to the various organs and clear differences were observed in the patterns of distribution among the organs examined. 4. The level of these isozymes was compared in serum of neonate and adult mice, and a higher level of isozyme I and a lower level of isozyme IV were found in neonates compared to adults. 5. These results suggest that each isozyme has different functional roles in individual organs and that these isozymes may be involved in proliferation and development of cells.     

华南兔组织乳酸脱氢酶同工酶的研究

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。     

AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle.

The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K₂L₂ and K₃M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K₄ also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M₄. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.     

Distribution of AMP-deaminase isozymes in rat tissues

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.     

Nonidentical subunits of human erythrocyte phosphofructokinase

Human erythrocyte and muscle phosphofructokinase (PFK) were purified completely by improved procedures. SDS-acrylamide gel electrophoresis in a discontinuous buffer system revealed two subunits (R and M) of erythrocyte PFK, the slower one (M) corresponding to the single subunit of muscle PFK. The staining intensity ratio R:M of the two bands of erythrocyte PFK was 2:1 or less. This suggests that native erythrocyte PFK contains multiple isoenzymes with different proportions of R and M, some being lost during purification. Nevertheless, isoelectric focusing showed single peaks of erythrocyte PFK (pI 5.0) and muscle PFK (pI 6.6), perhaps because of aggregation of erythrocyte PFK isoenzymes. Erythrocyte PFK from a patient with muscle PFK deficiency had a pI of 4.6 and could not be precipitated by antiserum against muscle PFK, findings compatible with the putative structure R4.     

Phosphofructokinase isozyme expression during myoblast differentiation

Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.     

臭鼩血清蛋白和六种组织乳酸脱氢酶同工酶的研究

本实验对臭鼩的血清蛋白及心肌、骨骼肌、肾脏、脾脏、肝脏,睾丸6种组织器官的乳酸脱氢酶(LDH)同工酶进行了聚丙烯酰胺凝胶盘状电泳的分析研究。臭鼩血清蛋白存在15—17条带,各组织的LDH同工酶均由5条带构成,其中心肌LDH-1、LDH-2和肾脏LDH-1各出现1条亚带。     

Pyruvate kinase isozymes in man

Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.     

Liver specific pyruvate kinase in pheasants Phasianus colchicus

While in chickens, the existence of a liver form of pyruvate kinase is controversial, the liver form of pyruvate kinase in pheasants, murres and puffins is electrophoretically distinct from that in muscle, brain, kidney, lung and small intestine. Although the forms in lungs, muscle, heart, brain and small intestine could not be reliably separated by electrophoresis, the functional characteristics of the lung and muscle forms of pyruvate kinase in the pheasant are distinct and can be classified as K and M isozymes respectively. Our data suggest that these birds possess at least three distinct isozymes of pyruvate kinase.     

Carbohydrate metabolism in temporal and persistent hypoglycemic chickens induced by insulin infusion

In order to elucidate the regulatory mechanism of blood glucose concentrations specific to chickens, carbohydrate metabolism in the liver, muscle and kidney and metabolite concentrations in the blood were investigated in chickens with acute and persistent hypoglycemia. Acute and persistent hypoglycemia were experimentally induced by a single injection of insulin (8 U/kg BW) or by continuous infusion of insulin (22.5 U/kg BW/day) for 4 days. Non-esterified fatty acid (NEFA) concentration in plasma and D-3-hydroxybutyrate (3HB) concentrations in liver and muscle increased in the acute hypoglycemia. Plasma NEFA concentration and 3HB concentration in the blood and liver were not changed at day 3 of persistent hypoglycemia, while 3HB concentration in the muscle was decreased. Phosphofructokinase (PFK) activity in the liver tended to increase but PFK and pyruvate kinase (PK) activities were unchanged in acute hypoglycemia. In persistent hypoglycemia, increase of hepatic PFK activity at day 1 in which it was reversed at day 3, and a small increase of muscle PK activity were observed, while PK and phosphoenolpyruvate carboxykinase (PEPCK) activities in the liver and kidney were not significantly changed. These results show that in the persistent hypoglycemic chickens, hepatic glycolysis transiently increases, which is followed by a small decrease, while glycolysis in muscles and gluconeogenesis in the liver and kidney are not significantly changed.     

金黄地鼠不同组织碱性磷酸酶同工酶研究

采用聚丙烯酰胺圆盘电泳方法,分析了金黄地鼠血清,肝脏,心脏和骨骼肌4种组织的碱性磷酸酶同工酶。结果表明,在同种动物中不同组织的碱性磷酸酶谱具有明显的组织特异性,并且各部门酶带有重叠现象。在不同组织中碱性磷酸酶具有特异性,这是与各组织的生理功能相适应的结果。     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

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.     

Survey of fructose 1,6-bisphosphatase isoenzyme in rat organs and ontogenic expression of the enzyme in rat fetus

1. Among eleven tissues of rat, the liver type of fructose 1,6-bisphosphatase (FBPase) subunit was detected in the liver, kidney, testis, pancreas and lung by Western blot analysis using anti-(liver FBPase) or anti-(muscle FBPase) serum. 2. The muscle type of the enzyme subunit was detected only in the pancreas other than skeletal muscle. Both types of the enzyme subunit were found in the pancreas. 3. Neither anti-(liver FBPase) nor anti-(muscle FBPase) serum detected the band of enzyme subunit on the blots of the extracts of brain, heart, small intestinal mucosa, spleen and placenta. 4. FBPase is present in fetal rat liver at least as early as the 14th day of gestation. 5. In agreement with the increase in immunological staining density, the level of the enzyme activity in fetal liver increased exponentially during fetal development. 6. The muscle enzyme was not detected until the fetus reached the 19th day of gestation.     

Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII.

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.