M<sub>2</sub> isozyme of pyruvate kinase from human kidney as the product of a separate gene: its purification and characterization

Harkins, Richard N.; Black, John; Rittenberg, Marvin B.

doi:10.1021/bi00636a018

Cited by 41 publications

(12 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This enzyme displays a slightly sigmoid satura tion curve with respect to PEP which is con verted to a hyperbolic curve in the presence of F-1,6-DP. In many papers it has been shown that M2-PK possesses intermediate regulatory properties between those of R-PK and Mi-PK [2,4,5,27]. In agreement with these studies, we have shown that M2-PK has a higher affinity for the substrate PEP than that of R-PK.…”

Section: Kinetic Studiessupporting

confidence: 90%

“…Mi-PK is present in muscle and brain and M2-PK in most other adult tissues (leukocytes in particular), foetal tissues and various tumours [1,2]. Mi-PK and M2-PK share a common antigenic reactivity [3,4], However, they display dif ferent kinetic properties with respect to phosphoenol pyruvate (PEP). Mi-PK dis-PK and M2-PK like L-PK and R-PK are encoded by the same structural gene, but translated from different mRNAs [13].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Kechemir

Max-Audit²,

Rosa³

1989

Enzyme

View full text Add to dashboard Cite

M(2)-type pyruvate kinases (M(2)-PK) have been isolated from human leukocytes and from the erythrocytes of a patient with erythrocyte PK hyperactivity. The kinetic characteristics of the patient erythrocyte M(2)-PK were similar to those of leukocyte M(2)-PK except for the Hill coefficient of phosphoenol pyruvate kinetics that showed little difference in the values. The patient erythrocyte M(2)-PK displayed complete immunological identity with leukocyte M(2)-PK in immunodiffusion, immunoblotting and immunoneutralization. The sensitivity to proteolysis by trypsin and the electrophoretic migration in different conditions were similar for the M(2)-PK of both origins. These results suggest an identity between this M(2)-PK abnormally present in erythrocytes and the M(2)-PK from leukocytes.

show abstract

Section: Kinetic Studiessupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Kechemir

Max-Audit²,

Rosa³

1989

Enzyme

View full text Add to dashboard Cite

show abstract

“…Whether M and K subunits, nant PYmvate kinase form and synthesis of all three types of which are immunologically indistinguishable, are encoded or not ~hosphofructokinase subunits. These patterns are not profoundly by separate genes is still in dispute (6,7,17,21). For phospho-different from those observed in either adult or fetal kidney (Table fructokinase, we and others have shown that it is encoded by three 1 ).…”

mentioning

confidence: 64%

Pyruvate Kinase and Phosphofructokinase Isozymes in Childhood Cancers

et al. 1982

View full text Add to dashboard Cite

“…As a result, our estimates of content are in error by the differences in mass between the two purified enzymes. Other workers have reported molecular masses for both Ml and M2 isozymes of 210,000-250,000 daltons (19,(24)(25)(26)(27). Rabbit muscle PyKi and PyKi precipitated from supernates of L2 by antibody covalently linked to acrylamide beads had identical mobility when examined by SDS-polyacrylamide gel electrophoresis; therefore, errors resulting from differences in molecular mass must be quite small.…”

Section: Resultsmentioning

confidence: 98%

Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

Hance¹,

Robin²,

Simon³

et al. 1980

J. Clin. Invest.

View full text Add to dashboard Cite

A B S T R A C T Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11+0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 ±0.04 U/,ug DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +0. 13; hypoxia, 0.94±0.13 ,g enzyme protein/,Lg DNA). Specific activity was not significantly different after 96h (L2: normoxia, 261±11; hypoxia, 261+14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic 02 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to characterizing enzymes, as well as quantitating cellular enzyme content. INTRODUCTIONChronic 02 depletion is an important clinical problem, but the cellular mechanisms responsible for adaptation to chronic hypoxia are obscure. The increased rate of glycolysis following acute hypoxic exposure was first described by Pasteur [3][4][5]. This stimulation ofglycolysis by chronic hypoxic exposure may also explain the bioenergetic differences observed between pulmonary artery endothelial cells (Po2 = 40 torr) and aortic endothelial cells (Po2 = 100 torr), as well as the differences observed in fetal and neonatal brain (6,7). Increased activity of rate-limiting glycolytic enzymes has also been found in fibroblast, endothelial, and kidney cell lines cultured under hypoxic conditions. Therefore, the phenomenon appears to be a common cellular response to 02 depletion. To investigate the mechanism by which the activity of PyKi is increased following chronic hypoxic exposure, we determined the isozyme type and content of PyKi present in a cloned cell line derived from rat lung (L2) and in WI-38 fibroblasts following hypoxic or nonnoxic cultivation. The results show an increase in the intracellular content of PyKi, with no significant change in specific activity (PyKi activity/PyKi content) or isozyme type present after hypoxic cultivation. This suggests that the availability of molecular 02 modulates the content of key glycolytic enzymes, which in turn regulate the generation of ATP through glycolysis. METHODSCell culture. L2 clone derived from rat lung was obtained from Dr. William Douglas in 36th population doubling (8) and maintained in cell culture by using Ham's F-12 medium containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 ,ug/ml streptomycin. Confluent plates were trypsinized and replated weekly with a 1:4 split, and experiments were performed on cells between the 40th and 55th population doubling.WI-38 fibroblasts were obtained from the American Type Culture Collect...

show abstract

M₂ isozyme of pyruvate kinase from human kidney as the product of a separate gene: its purification and characterization

Cited by 41 publications

References 36 publications

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Pyruvate Kinase and Phosphofructokinase Isozymes in Childhood Cancers

Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

Contact Info

Product

Resources

About

M2 isozyme of pyruvate kinase from human kidney as the product of a separate gene: its purification and characterization

Cited by 41 publications

References 36 publications

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Comparative Study of Human M2-Type Pyruvate Kinases Isolated from Human Leukocytes and Erythrocytes of a Patient with Red Cell Pyruvate Kinase Hyperactivity

Pyruvate Kinase and Phosphofructokinase Isozymes in Childhood Cancers

Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

Contact Info

Product

Resources

About

M₂ isozyme of pyruvate kinase from human kidney as the product of a separate gene: its purification and characterization