José Carreras

Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase, creatine kinase and enolase activity and isoenzymes in breast carcinoma

We have compared the levels of phosphoglycerate mutase (EC 5.4.2.1), 2,3-bisphosphoglycerate phosphatase (EC 3.1.3.13), creatine kinase (EC 2.7.3.2) and enolase (EC 4.2.1.11) activities and the distribution of their isoenzymes in normal breast tissue and in breast carcinoma. Tumour tissue had higher phosphoglycerate mutase and enolase activity than normal tissue. Creatine kinase activity was higher in seven out of 12 tumours. In contrast 2,3-bisphosphoglycerate phosphatase activity was lower. Phosphoglycerate mutase, enolase and 2,3-bisphosphoglycerate phosphatase presented greater changes in the oestrogen receptor-negative/progesterone receptor-negative breast carcinomas than in the steroid receptor-positive tumours. Determined by electrophoresis, type BB phosphoglycerate mutase, type BB creatine kinase and αα-enolase were the major isoenzymes detected in normal breast tissue. Types αγ and γγ enolase, types MB and MM phosphoglycerate mutase were detected in much lower proportions. In tumours a decrease of phosphoglycerate mutase isoenzymes possessing M-type subunit and some increase of enolase isoenzymes possessing γ-type subunit was observed. No detectable change was observed in the creatine kinase phenotype.

Effects of Thyroid Hormone on mRNAs of Phosphoglycerate Mutase Subunits in Rat Muscle during Development

Background: We previously showed that triiodothyronine (T3) stimulates muscle phosphoglycerate mutase (PGAM) activity and isozyme transition in rat skeletal and cardiac muscles. Methods: The effects of T3 on PGAM types B and M subunit expression in rat muscle during development are reported. Results: T3 administration during the first 21 days of rat life more than doubles type M PGAM mRNA levels, but produces minor effects on type B PGAM mRNA levels. The antihormone propylthiouracil (PTU) slightly decreases both type B and M mRNA levels, but this decrease is not statistically significant. Conclusion: Thyroid hormone influences PGAM mRNA isozyme levels differently and increases type M mRNA.

Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phospho glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P2 phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.

Vanadate inhibits 2,3-bisphosphoglycerate dependent phosphoglycerate mutases but does not affect the 2,3-bisphosphoglycerate independent phosphoglycerate mutases

Abstract There are two types of phosphoglycerate mutases. The 2,3-bisphosphoglycerate dependent phosphoglycerate mutases are inhibited by vanadate. In contrast, the 2,3-bisphosphoglycerate independent mutases are not affected. The effect of vanadate varies with pH, and can be reversed by dilution, EDTA and norepinephrine. The differential effect of vanadate on the two types of phosphoglycerate mutases supplies a novel way to easily differentiate both types of enzymes. In addition, it may contribute to the clarification of the mechanism of action of the 2,3-bisphosphoglycerate independent phosphoglycerate mutases.

Phylogeny and ontogeny of the phosphoglycerate mutases—IV. Distribution of glycerate-2,3-P2 dependent and independent phosphoglycerate mutases in algae, fungi, plants and animals

1. The distribution of the glycerate-2,3-P2 dependent and independent phosphoglycerate mutases (PGM) has been studied in more than eighty species. 2. PGM activity in the extracts has been measured in the presence and in the absence of glycerate-2,3-P2, at pH 7.5 and at pH 8.5. 3. All samples with glycerate-2,3-P2 dependent PGM possess higher activity at pH 7.5 than at pH 8.5. In contrast, samples with glycerate-2,3-P2 independent PGM possess lower activity at pH 7.5 than at pH 8.5. 4. In algae and fungi both glycerate-2,3-P2 dependent and independent PGM have been found. 5. In plants only glycerate-2,3-P2 independent PGM has been detected. 6. In animals both types of PGM are present. Independent PGM activity is present in sponges, coelenterates, myriapods, arachnids and echinoderms. Glycerate-2,3-P2 dependent PGM is present in platyhelminths, mollusks, annelids, crustaceans, insects and vertebrates.

Phylogeny and ontogeny of the phosphoglycerate mutases—II. Characterization of phosphoglycerate mutase isozymes from vertebrates by their thermal lability and sensitivity to the sulfhydryl group reagents

Abstract 1. 1. To characterize the three phosphoglycerate mutase (PGM) isozymes present in vertebrates (types M, B and MB) their sensitivity to the reagents of the sulfhydryl groups and to heat treatment has been studied. 2. 2. In mammals and reptiles type M PGM is not affected by the —SH group reagents, type MB PGM is inhibited about 50% and type B PGM is fully inhibited. Types B and MB PGM show greater heat lability than type M PGM. 3. 3. In amphibians and fishes PGM isozymes do not differ in their sensitivity to the —SH reagents. 4. 4. The results strongly support the homodimeric and heterodimeric structure suggested for PGM isozymes and favour their genetic origin.

Phylogeny and ontogeny of the phosphoglycerate mutases—I. Electrophoretic phenotypes of the glycerate-2,3-P2 dependent phosphoglycerate mutase in vertebrates

Abstract 1. 1. The electrophoretic phenotype of phosphoglycerate mutase in tissues from different classes of vertebrates at several stages of development have been analyzed on cellulose acetate. 2. 2. Mammals, reptiles, amphibians and fish show a common three-banded isozyme pattern. 3. 3. The three isozymes vary in their relative distribution from tissue to tissue and during growth. 4. 4. In birds electrophoretically distinguishable phosphoglycerate mutase isozymes have not been detected. 5. 5. The results support a genetic basis for the phosphoglycerate mutase isozymes and suggest that gene duplication may have occurred early in vertebrate evolution.

Regulation of muscle phosphofructokinase by physiological concentrations of bisphosphorylated hexoses: Effect of alkalinization

To clarify the role of glucose-1,6-P2, fructose-2,6-P2 and fructose-1,6-P2 in the control of the glycolytic flux during muscle contraction, we have determined the activity of muscle phosphofructokinase in the presence of physiological concentrations of these bisphosphorylated hexoses and other allosteric effectors, and at increasing pH values. In the presence of fructose-2,6-P2, both glucose-1,6-P2 and fructose-1,6-P2 can additionally activate the enzyme and partially counteract citrate inhibition. Activation of phosphofructokinase produced by alkalinization increases in the presence of the bisphosphorylated hexoses. It is suggested that the hexose bisphosphates could play a significant role in the initial burst of the glycolytic flux during muscle contraction, when an alkaline pH shift is produced.

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα‐, αγ‐, and γγ‐enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα‐enolase was the predominant isoenzyme, and αγ‐enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα‐enolase and a lower proportion of γγ‐enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα‐enolase and lowest proportion of γγ‐enolase.

Effect of vanadate on the formation and stability of the phosphoenzyme forms of 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and of phosphoglucomutase

2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-bisphospho-D-glycerate:2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) and phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha -D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1), which are markedly inhibited by vanadate, possess a ping-pong mechanism involving an intermediate phosphoenzyme. The formation and the stability of these phosphoenzymes have been examined spectrophotometrically in the absence of vanadate. Vanadate does not inhibit the phosphorylation of either mutase by its cofactor. The instability of the phosphoenzyme form of phosphoglycerate mutase increases in the presence of vanadate, but the stability of the phosphorylated phosphoglucomutase is not affected.