Archivos de biologia y medicina experimentales最新文献

We have studied the cell differentiation of Trypanosoma cruzi in an vitro system that allows the transformation of epimastigotes into metacyclic trypomastigotes. Intracellular cAMP levels of epimastigotes increased 3 fold prior to their differentiation into metacyclics where cAMP remained elevated 3.7 fold with respect to epimastigotes. We also observed a 3 fold increase in the specific activity of cAMP-binding of metacyclics crude homogenates. This activity resided in a cAMP-binding receptor protein (CARPT) which was different from the typical cAMP-binding subunits (RI and RII) of cAMP-dependent protein kinases, as shown by the use of polyclonal antibodies prepared against these two types of proteins. Anti-RI antibodies did not react with CARPT, and anti-RII antibodies gave a cross reaction with CARPT which was at least 1,000 fold less sensitive than the one shown by the homologous antigen. On Western blots CARPT displayed a major band with Mr = 87,000 instead of Mr = 56,000 for RII. These studies implicate that cAMP may act as a mediator of the cell differentiation of T. cruzi by a mechanism involving a novel type of cAMP-binding receptor.

Cyclin/PCNA (auxiliary protein of DNA polymerase delta) is a proliferation sensitive DNA replication protein whose synthesis is modulated during the cell cycle. All available information suggests that cyclin/PCNA may trigger DNA replication following the formation of pre-replicative complexes at the G1/S transition border.

Several studies have been performed on the structure of muscle pyruvate kinase. X-ray diffraction has provided a three-dimensional picture of the active site, and chemical modification studies have revealed essential amino acid residues for substrate binding or catalysis. We have shown that 8-azido-ADP (N3 ADP) behaves as a photoaffinity label for the enzyme. This reagent upon irradiation produces inactivation of the enzyme, and the activity loss is protected by nucleotides. The partially modified enzyme shows the same Km for ADP as the native one suggesting an "all or none" inactivation effect. The incorporation of 1 mole of 14C-N3 ADP per subunit correlates with complete inactivation. A radioactive peptide was isolated from the enzyme labeled with 14C-N3 ADP. The partial sequence of this peptide showed that it corresponds to the same peptide isolated from rabbit muscle pyruvate kinase labeled with dialdehyde-ADP and with trinitrobenzenesulfonate. This peptide is identical to a region in the cat and chicken muscle enzymes, and also a high degree of homology is found in a region of the rat liver and yeast enzymes. These studies show that N3 ADP binds to the same site as dialdehyde-ADP in rabbit muscle pyruvate kinase, and this site seems to be the nucleotide binding site.

The rate of degradation of radioactive labeled mitochondrial proteins synthesized both in vitro and in vivo by isolated yeast mitochondria and growing yeast cells respectively, has been studied. It was found that the in vitro-synthesized mitochondrial proteins are rapidly degraded by an energy-dependent proteolytic system. Under the same experimental conditions the in vivo-synthesized mitochondrial proteins are slowly degraded to a limited extent by a protease which is slightly inhibited by ATP. During this period, the mitochondria are coupled and metabolically active. It is proposed that mitochondria possess an energy-dependent proteolytic system that recognizes as substrates either "abnormal" proteins or unassembled protein subunits encoded in the mitochondrial genome. An apparently different system, which is independent of energy, seems to be responsible for the slow and limited degradation of "normal" mitochondrial proteins.

Bacillus cereus has proved to be one of the most interesting microorganisms in the study of beta-lactamases. It secrets these enzymes very efficiently and, frequently, in multiple forms. Three different forms are produced by strain 569/H; mutant 5/B of the same microorganism is constitutive for the secretion of beta-lactamases I and II. The present study, based on secondary structure prediction by two independent methods, states the relationship among the structures of beta-lactamases I, II and III produced by B. cereus 569/H and beta-lactamase I from the strain 5/B of this microorganism. A strong similarity is also established for the enzyme type III of B. cereus and the enzyme type I produced by B. licheniformis which could have an evolutionary explanation. A structural analysis of the leader peptide regions of these enzymes by the method of Mohana and Argos is also reported.