RESUMO
The GA733-2 antigen (GA733) is a homotypic calcium-independent cell adhesion molecule (CAM) present in most normal human epithelial cells and gastrointestinal carcinomas. Because oligomerization of some CAMs regulates cell adhesion and signal transduction, the correlation between GA733 oligomeric state and cell-cell adhesion was investigated. Sedimentation equilibrium studies showed that full-length (-FL) GA733 exists as dimers and tetramers in solution, whereas the GA733 extracellular domain (-EC) is a monomer. The Kd of GA733-FL is less than 10 nm for the monomer-dimer association, whereas the dimer-tetramer association is about 1000-fold weaker (Kd approximately 10 microm). Chemical cross-linking of purified GA733-FL in solution resulted in a major product corresponding to GA733 dimers, and minor amounts of trimers and tetramers. However, GA733-EC cross-linked under the same conditions was consistently a monomer. Chemical cross-linking of dissociated colon carcinoma cells produced predominantly GA733 dimers, whereas cross-linking of cells in monolayers yielded some tetramers as well. GA733-FL retained its cell-cell adhesion function as shown by inhibition of cell aggregation, whereas monomeric GA733-EC was inactive. These data show that GA733 exists predominantly as high affinity noncovalent cis-dimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is most likely the head-to-head interaction between GA733 cis-dimers on opposing cells that represents its cell-cell adhesion activity.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Neoplasias do Colo/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/fisiologia , Biopolímeros , Western Blotting , Células CACO-2 , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/patologia , Eletroforese em Gel de Poliacrilamida , Molécula de Adesão da Célula Epitelial , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence.
Assuntos
Proteínas de Bactérias/genética , Ferroquelatase/genética , Genes Bacterianos , Rhodobacter capsulatus/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Rhodobacter capsulatus/enzimologiaRESUMO
In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.
