Isoenzyme Type Research Articles

Isolation and distribution of myosin isoenzymes in chicken pectoralis muscle

The preparation of rabbit antibodies uniquely specific for the alkali 1 (A1) and alkali 2 (A2) light chains of chicken pectoralis myosin has led to the direct isolation of two homodimeric species of myosin: A1-myosin and A2-myosin, molecules which contain the same light chain on each head. The existence of a heterodimeric species, containing both A1 and A2 light chains, was also inferred. The three types of alkali light chain isoenzymes occur in approximately equal amounts in adult chicken pectoralis muscle. The specificities of the antibodies were determined by modified Farr and solid phase radioimmunoassays, as well as by antibody-affinity chromatography. The determinants in myosin that are recognized by the purified antibodies appear to be confined to the N-terminal sequences of the alkali light chains. As a result of this narrow specificity, these immunological reagents can be used to characterize the distribution of A1 and A2 within the myosin molecule, and to localize the individual light chains within the muscle. By labeling the antibodies with a fluorescent marker we have shown that A1 and A2 are present within each myofibril, as well as within the same fiber (Lowey et al., 1979 a). Moreover, by using goat anti-rabbit immunoglobulin to enhance the visualization of the primary antibodies against the light chains, we have demonstrated in the electron microscope that A1 and A2 co-exist along the length of each myofilament. This observation suggests that whatever functional differences may exist among the alkali light chain isoenzymes, they must operate within the constraints of a single filament.

Studies on alkaline phosphatase isoenzyme in gastric carcinoma tissues.

An analysis of the alkaline phosphatase (ALP) isoenzyme in 75 gastric carcinoma tissues was performed. The properties and the originating cells of ALP isoenzymes in them were clarified by using both enzyme histochemical and biochemical methods which had respective superiorities. Also the relationship between intestinal metaplasia and gastric carcinoma was discussed. Liver, placental and small intestinal type ALPs were found in the carcinoma tissues. No ALP activity was found in the carcinoma cells except in the stromal cells in 46 out of 75 cases. In all of these cases, only liver type ALP was found. While, in 29 cases, the activity was also found in carcinoma cells. In these cases, placental and small intestinal type ALPs were found in addition to liver type ALP in 26 and 3 cases, respectively These results indicated that liver type ALP came from the stromal cells and placental and small intestinal type ALPs came from carcinoma cells. The ALP found in carcinoma cells was placental (Nagao isoenzyme) type in most cases. While, the ALP in intestinal metaplastic epithelial cells was small intestinal type in all cases. No sufficient evidence indicating the relationship between intestinal metaplasia and gastric carcinoma was obtained in respect of ALP isoenzyme.

Comparison of the subunit compositions and ATPase activities of myosin in the myocardium and conduction system

The light chains and ATPase activities of specialized and ordinary muscle myosin from bovine heart were compared. The specialized heart muscle was composed of the A-V node, bundle of His, and right and left bundle branches. The yield of myosin from the specialized heart muscle was approximately 20% of that from the ordinary heart muscle. On sodium dodecyl sulfate (SDS)-polyacrylamide gel (10%) electrophoresis, ordinary heart myosin gave two bands of low molecular weight (light chains) as reported previously, whereas specialized heart myosin gave three bands. By comparison of the electrophoretic mobilities of these light chains with those of proteins of known molecular weight, the molecular weights of the two myosin light chains from ordinary heart muscle were estimated as 25 000 (LC 1) and 18 000 (LC 2), and those of the three bands of specialized heart myosin as 25 000 (LC 1), 22 500 (LC 1′) and 18 000 (LC 2). There was approximately 3.5 mol of myosin light chains/mol of myosin in both tissue (Ordinary heart muscle: LC 1 1.7 mol, LC 2 1.8 mol; Specialized heart muscle: LC 1 0.9 mol, LC 1′ 1.1 mol, LC 2 1.5 mol). The specific activities of myosin K +-EDTA- and Ca 2+-activated ATPase ( V max values in μmol phosphate/mg/min) in the two types of heart myosin were similar. These results suggest the existence of a new type of cardiac myosin isoenzyme in specialized heart muscle.

Aerobic glycolysis of tumor cells

A high aerobic glycolysis (aerobic lactate production) is the most significant feature of the energy metabolism of rapidly growing tumor cells. Several mechanisms, which may be different in different cell lines, seem to be involved in this characteristic of energy metabolism of the tumor cell. Changes in the cell membrane leading to increased uptake and utilization of glucose, a high level of fetal types of isoenzymes, a decreased number of mitochondria and a reduced capacity to metabolize pyruvate are some factors which must be taken into consideration. It is not possible to favour one of them at the present time.

Effect of thyroid hormone on the vitamin D metabolism and bone pathology of adult female wistar rats

1. 1. Groups of young and mature female Wistar rats were made thyrotoxic by subcutaneous injections of T 4 or T 3. Plasma and femurs were analysed for chemical parameters as indices of altered bone pathology. 2. 2. Thyrotoxic animals showed a significant decrease in the level of albumin in plasma and an increase in alkaline phosphatase. Electrophoresis of alkaline phosphatase suggested an increase in bone type isoenzyme. 3. 3. Young and mature rats treated with T 3 showed changes in bone analysis with a significant rise in the water content and a decrease in calcium. 4. 4. Young thyrotoxic rats had the same plasma 25-hydroxyvitamin D (25[OH]D) as controls and there was no change in the in vivo conversion of vitamin D to 25(OH)D. However, mature rats treated with T 3 had a slightly but significantly lower in vivo conversion of 25(OH)D to more polar derivatives. 5. 5. These results give little support to the suggestions that thyroid induced changes in bone are due to a direct effect on vitamin D metabolism.

Change in alkaline phosphatase isoenzyme pattern in urine as possible marker for renal disease

The human kidney contains two types of alkaline phosphatase (AP) isoenzymes: a hepatic type of AP and an intestinal-like AP. Intestinal-like AP, measured by immunotitration techniques, is a minor component (1 to 4%) of the total AP activity. It is found only in the particle-free fraction (cytoplasm) and is located, with immunofluorescent techniques, in some of the proximal convoluted tubules. Urinary AP activity is found after high-speed centrifugation in the supernatant (x 100,000g), as well as in the sediment, and may be extracted from the sediment after solubilization with n-butanol. Both types of these renal isoenzymes contribute to urinary AP activity. Biochemical characterization (effect of inhibitors, thermostability, denaturing with urea, and so on) revealed that urinary intestinal-like AP and renal intestinal-like AP are identical. Both, however, have been distinguished as multiple forms of AP from the small intestine. Most of the urinary AP activity of healthy persons (22 volunteers) was found in the sediment and consisted of liver-type AP. Urinary AP of patients with diseases, after application of potentially nephrotoxic drugs or during rejection episodes of renal allografts, contains little sediment activity, but it contains increased amounts of urinary intestinal-like AP.

Aldolase Isoenzyme Patterns during Human Ontogeny and in Lung, Kidney and Breast Cancer

Enzyme patterns characteristic of fetal tissue have been noted in some experimental tumor models, particularly in hepatomas. In this study we undertook to determine whether biochemical evidence of a similar reversion could be detected in tumors of other human organs. As marker, we chose to use the aldolase isoenzymes A, B and C, for which distinct adult and fetal tissue patterns have been described. Using monospecific antibodies, we determined the aldolase isoenzyme pattern in a variety of human organs ranging in age from 14 to 40 weeks of gestation, in the 2- to 3-month postnatal period and in adults. In addition, 19 breast cancers, 19 primary lung cancers and 8 kidney cancers were examined. Our studies on breast cancer revealed three apparently distinct groups -- one showing primarily the A isoenzyme type (6 cases), a second containing mainly A with considerable quantities of B and C isoenzymes (9 cases) and a third group (4 cases) which may contain a different isoenzyme altogether since the combined activity of the three known forms was less than 100% in each case. In lung cancer, fetal characteristics could be substantiated since in fetal and adult lung tissue, the isoenzyme pattern is almost identical; 3 out of 19 cases showed substantial quantities of the B isoenzyme. In kidney tumors, a reversion to the A form with an appreciable fraction of the C form was found, which is similar to the fetal pattern.

Regulatory properties of pyruvate kinase muscle of the crayfish orconectes limosus raf. (crustacea: decapoda)

1.1. Electrophoretic separation of extracts from O. limosus crayfish abdominal muscle showed the existence of one form of pyruvate kinase which was moving towards the anode faster than L type isoenzyme from rat liver.2.2. After electrophoresis pyruvate kinase from crayfish muscle showed sigmoidal kinetics with phosphoenolpyruvate as substrate. Fructose-1,6-diphosphate activated the enzyme allosterically. The results suggest that pyruvate kinase from crustacean muscle can exist in interconvertible forms having a different affinity for the substrate.

Myosin isoenzyme redistribution in chronic heart overload.

Since the first observation by Spann et al., it has become clear that in cardiac hypertrophy induced by a mechanical overloading, the velocity of shortening of the cardiac muscle (Vmax) is reduced (see ref. 2 for review). Most authors agree that this mechanical alteration is accompanied by a decrease in the Ca2+-dependent ATPase activity of myosin (see ref. 3 for review). The molecular basis of such changes was unknown because the structural modifications of the myosin molecule were ill-defined. Nevertheless, it has recently been shown that, like skeletal muscle myosin, cardiac myosin is composed of several polymorphic forms, comparable to isoenzymes. In the skeletal muscle, new functional requirements can induce changes in both contractile activity and type of myosin isoenzyme synthesised. We now report that an increase in cardiac work produced by mechanical overloading in rats induces the preferential synthesis of a cardiac myosin isoenzyme characterised by specific immunological and electrophoretic properties and exhibiting a lower ATPase activity. This adaptive change could account for the reduced shortening speed of this hypertrophied cardiac muscle.

The effect of sucrose and other carbohydrates on human alkaline phosphatase isoenzyme activity

The modification of alkaline phosphatase (ALP) activity by ten carbohydrates ( d-isomers) was studied. These included three disaccharides : sucrose, lactose and maltose; four hexoses: glucose, galactose, mannose and fructose; and three pentoses: arabinose, ribose and xylose. In all cases the inhibition of placental isoenzyme activity was in proportion to the concentration of carbohydrate. Intestinal and hepatic ALP was activated at low carbohydrate concentrations and inhibited at high concentration. The exception was maltose which inhibited hepatic alkaline phosphatase markedly at all concentrations. Lineweaver-Burk plots generated by kinetic studies at pH 9.8, 10.15, 10.5 and 10.7 showed a mixed-type inhibition. With the use of sucrose in the assay medium it was possible to distinguish two types of term placental-like ALP isoenzymes of tumor origin, which differ in their sensitivity to inhibition by l-leucine. The Nagao type ( d-leucine sensitive) was inhibited more by sucrose than was the non- d-leucine sensitive isoenzyme. The inhibitory effect of carbohydrates on placental ALP activity was independent of pH and substrate concentration, unlike the modifying effect on the activity of intestinal and hepatic enzyme by the carbohydrates studied. It is suggested that carbohydrate-rich media may accentuate conformational differences of the various isoenzymes with consequent effects on the availability of the active sites to the substrate.

Changes of the cAMP-Dependent Protein Kinases during Differentiation of Lens Cells

Several changes occur in the properties of cAMP-dependent protein kinase during the differentiation of lens epithelial cells, and/or in the course of aging of fiber cells. Two types of isoenzymes (I a

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.

Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways.

Etude des isoenzymes de l'amylase dans une tumeur pulmonaire secretante d'amylase

The plasma and pulmonary tumour are studied in a patient with lung metastasis. An abnormal and neuraminidase-sensible isoenzyme is noted both in plasma and tumour. Homogenates obtained from the tumour by two different mechanic disruption procedures denote an easier liberation for the salivary type isoenzymes than for pancreatic types. It is suggested that the salivary enzymes are synthesized in the cytoplasm during many inflammatory processes and secreted without cellular necrosis.

Chronic Myelocytic Leukemia

In three patients with chronic myelocytic leukemia who were heterozygous at the X-linked glucose-6-phospháte dehydrogenase locus, lymphocytes were studied to determine if they had the same stem cell origin as the leukemic myeloid cells. Normal tissues such as skin had both B and A glucose-6-phosphate dehydrogenase isoenzymes, but the leukemic myelogenous cells displayed only one isoenzyme type, consistent with their clonal origin. A population of cells with undoubted thymus-derived (T)-lymphocyte characteristics had both isoenzymes. Presumably, then, these T cells did not arise from the leukemic stem cell, either because they antedated the development of leukemia in that stem cell or, more likely, because they arose from progenitors not involved by the disease. In contrast, another population of lymphocytes showed only one isoenzyme type, suggesting that it arose from the chronic myelocytic leukemia stem cell. However, although this population contained many cells with the characteristics of bone marrow-derived (B) lymphocytes, it is not certain that the single enzyme produced by the cells over all can be attributed to B lymphocytes rather than to contaminating non-B-lymphoid cells.

Pancreatitis-like isoamylase pattern in normal persons

On the assumption that a rise in the pancreatic type isoamylases may not necessarily indicate underlying pancreatitis, genetic studies of human serum and urinary amylase isoenzymes have been performed with the use of electrophoresis. Although the preponderant increase in the two principal pancreatic isoamylases Amylase-1 and 2 has been accepted to be a specific index of pancreatic involvement, 1.68% of normal persons had Amylase-2 with an elevated amylase activity (named “Dominant Amylase-2”) up to the same levels as the major isoenzymes. Results of pancreozymin-secretin test and other laboratory findings of these persons with Dominant Amylase-2 were all within normal ranges. Pedigree studies confirmed an autosomal dominant mode of inheritance for this variant. The importance of serial determination and pedigree investigations has been shown to distinguish normal persons having Dominant Amylase-2 from patients with pancreatitis without elevated amylase activity. The existence of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind before coming to a conclusion when amylase isoenzymes are used for clinical medicine, though preponderance of the pancreatic type isoenzymes in serum and urine has been revealed to be a characteristic finding in pancreatitis. Knowledge of amylase genetic polymorphism provides a scientific basis for amylase isoenzyme interpretation.

Clonal markers in the study of the origin and growth of human atherosclerotic lesions.

The X-linked enzyme, glucose-6-phosphate dehydrogenase (G-6-PD) was used as a cellular marker to study the clonal characteristics of human atherosclerotic lesions from females heterozygous for G-6-PD isoenzymes. Portions of uninvolved aortic wall contained both isoenzyme types (A and B), and their isoenzyme patterns were used to establish criteria for polyclonal lesions. Portions of uterine leiomyomas contained predominantly one isoenzyme type (either all A or all B) and their isoenzyme patterns were used to establish criteria for monoclonal lesions. These techniques were used to address three questions concerning atherogenesis. First, evidence for the monoclonal origin of fibrous-capped plaques was provided by the findings that small plaques had G-6-PD isoenzyme distributions similar to those of leimyomas; that in large plaques with multiple portions assayed for G-6-PD, a large proportion (25 of 26, 96%) of plaques had monoclonal characteristics; and that multiple monoclonal portions were present in the same plaque. Second, the role of the fatty streak as a precursor of fibrous plaques was supported by the demonstration that a proportion (11 of 66, 16.7%) of fatty streaks contained isoenzyme patterns intermediate between those of polyclonal uninvolved aortic wall and monoclonal leiomyomas. Increased cellularity of fatty streaks correlated with increased deviation of isoenzyme pattern toward monoclonality. Third, the assay of portions of both small and large plaques provided no evidence for clonal selection as plaques increase in size.

Clinical evaluation of the pancreatitis-like isoamylase pattern in normal persons.

Amylase isoenzyme analysis of serum and urine has been performed in 4001 normal persons and 500 patients with various disease using electrophoresis on thin layer polyacrylamide gel. Although elevation of amylase activity in amylase-1 and 2 has been reported to be the specific findings in patients with pancreatitis, 1.69% of normal persons had an elevated Amylase-2(named "Dominant Amylase-2") up to the same levels as major isoenzymes (Amylase-1 and 3), along with Amylase-1. Pedigree study confirmed an autosomal dominant mode of inheritance for Dominant Amylase-2. Knowledge of the genetic polymorphism is of importance in clinical assessment of amylase isoenzymes in patients having an elevated Amylase-2 suggestive of pancreatitis. Predominance of the pancreatic components in serum and urine has been revealed to be a specific index of pancreatic involvement. However, the existecne of an inherited trait of pancreatitis-like isoamylase pattern in healthy individuals must be borne in mind. On the basis of the present study, it may be concluded that a rise in the pancreatic type isoenzymes may not necessarily indicate underlying pancreatitis, especially in the absence of elevated amylase and lipase levels.

Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.

Myosin in primary cultures of hamster heart cells

Primary cultures of neonatal hamster heart cells contained a heterogeneous population of cell types. Muscle cells represented one such type, and could be identified by their ability to contract spontaneously in culture and by their morphology when examined directly by phase contrast microscopy or after staining with phosphotungstic acid-hematoxylin. Overgrowth of muscle cells by more rapidly dividing nonmuscle cells was prevented by treatment of the cultures with the mitotic inhibitor, 1-β- d-arabinofuranosylcytosine. The muscle cells increased their specific content of myosin over a period of 5 days in culture. They were then, however, relatively immature when compared with the predominating cells in 1-day-old hamster hearts. The structural and enzymatic properties of purified myosin from the cultures were not different from those of the isoenzyme type of this protein normally found in neonatal hamster hearts, and nonmuscle cells contributed little of the total myosin.