Location of adult and fetal aldolases A, B, and C by immunoperoxidase technique in LF fast-growing rat hepatomas.

The resurgence of aldolase isozymes in cancerous tissues is a well-known but poorly understood phenomenon. This resurgence poses the problem of whether or not adult and fetal aldolase isozymes are produced by the same cells. For clarification of this question, the immunoperoxidase technique was used to locate aldolases A, B, and C in one type of fast-growing hepatoma, the LF hepatoma and, by comparison, in normal adult liver. Under optical microscopy, aldolases A and C were located in the cytoplasm of almost all of the cancerous cells. An isozyme antigenically identical with aldolase B was also demonstrated to be present in almost all of the cells, but the reaction indicating the presence of this isozyme was weaker. In normal adult liver, only aldolases A and B were demonstrated to be present in almost all the hepatocytes. Under electron microscopy in LF hepatoma, the three isozymes were found to be present mainly in the cytoplasm. These facts suggest that the three types of aldolase are very probably present in the same cells at the same time, and they provide indirect arguments leading us to think that the resurgence of fetal aldolase isozymes in cancer is not the consequence of cellular selection but is due to a disturbance at the gene control level.

Purification and characterization of human erythrocyte uridylyl transferase.

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.

Aldolase B in the liver of senescent rats.

The process of enzymatic aging was studied in livers of adult and senescent rats for aldolase B. No "cross-reacting material" was found in livers of 27 to 30-month-old rats, estimated by the ratio aldolase activity-antigen amount. The activity towards the two substrates of aldolase, fructose 1,6-diphosphate and fructose 1-phosphate did not vary in senescent animals. Moreover, other physico-chemical properties of the enzyme such as thermal inactivation, immunological reactivity and Michaelis constant remain unchanged. These results provide arguments against the occurrence of errors in protein synthesis as a cause of aging.

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes.

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.

Pyridoxal 5' phosphate involvement in age-related modifications of tyrosine aminotransferase: multiple form patterns.

Multiple form patterns of tyrosine aminotransferase were studied in senescent and adult rat liver. Two main modifications were described for "old" rat enzyme: (i) appearance of a new molecular form, specific for old rats, eluted after adult form III and having other properties identical to this form; (ii) disappearance of intermediate forms II and III after enzyme induction; this result seems to be due to acceleration of the conversion process. Vitamin B6 deficiency of old rats explain this and other (previously described) post-translational modifications of "old" tyrosine aminotransferase. The influence of pyridoxal 5' phosphate and the role of protease(s) in the multiple form conversion are discussed. Moreover we show the possibility of a correlation between in vivo alterations of the enzyme molecule and modifications of tyrosine aminotransferase multiple form patterns observed in vitro.

Differentiation in vivo and in vitro of pyruvate kinase isozymes in rat muscle.

The M type isozymes of Pyruvate-kinase have been studied by isoelectrofocusing in thin layer acrylamide ampholine gel, during the ontogeny of rat muscle in vivo and during the differentiation in vitro of myoblasts of a line established by Yaffe. In both cases, multiple subbands have been seen; the most acid (pHi 5.2) was the predominant band in myoblasts and in fetal muscle at the 15th day. Several more cathodic bands appear sequentially in vitro as in vivo, one of them corresponding to the "M2" or "K" band (predominant in kidney). The most cathodic band M1 (pHi 7.3), characteristic of the adult muscle, appears at the 9th day of culture in vitro in multinucleated myotubes and at the 20th day of fetal life in vivo. Kinetic results confirm these electrofocusing results, showing in fetal muscle and in myoblasts a sigmoid saturation curve of pyruvate kinase activity with phosphoenolpyruvate as substrate. This allosteric kinetic is progressively replaced by a Michaëlian kinetics in vitro as in vivo. Consequently, the studies in vitro may serve as a model for myogenesis in vivo, and may contribute to the understanding of the significance of the multiple forms of pyruvate-kinase during this myogenesis.

Isoelectrofocusing of aldolase B from normal human livers and from livers with hereditary fructose intolerance.

By isoelectrofocusing in thin-layer acrylamide-ampholine gel, normal human aldolase B has been resolved into 5 bands. Moreover we were able to specifically stain (after isoelectrofocusing) the mutated aldolase B in livers with hereditary fructose intolerance, and to show that only the 3 most anodic bands are seen. Some different hypotheses are discussed to account for the microheterogeneity of the normal aldolase B, and for the different isoelectrofocusing pattern found in livers with hereditary fructose intolerance.

DNA methylation and gene expression.

In the field of studies of the mechanisms of gene expression, much recent work using the methods of genetic engineering, have suggested the hypothesis of a role of DNA methylation (on some cytosines) in the control of gene activity. Undermethylation is generally accompanied by gene expression. A review of studies on DNA methylation in some physiopathologic conditions (such as development and cancer) and of recent therapeutic trials in beta thalassemia, allows to conclude that there is a correlation between DNA methylation pattern and gene expression, rather than a direct causal effect.