RESUMO
It has been believed that isoamylase (ISA)-type α-glucan debranching enzymes (DBEs) play crucial roles not only in α-glucan degradation but also in the biosynthesis by affecting the structure of glucans, although molecular basis on distinct roles of the individual DBEs has not fully understood. In an attempt to relate the roles of DBEs to their chain-length specificities, we analyzed the chain-length distribution of DBE enzymatic reaction products by using purified DBEs from various sources including rice, cyanobacteria, and bacteria. When DBEs were incubated with phytoglycogen, their chain-length specificities were divided into three groups. First, rice endosperm ISA3 (OsISA3) and Eschericia coli GlgX (EcoGlgX) almost exclusively debranched chains having degree of polymerization (DP) of 3 and 4. Second, OsISA1, Pseudomonas amyloderamosa ISA (PsaISA), and rice pullulanase (OsPUL) could debranch a wide range of chains of DPâ§3. Third, both cyanobacteria ISAs, Cyanothece ATCC 51142 ISA (CytISA) and Synechococcus elongatus PCC7942 ISA (ScoISA), showed the intermediate chain-length preference, because they removed chains of mainly DP3-4 and DP3-6, respectively, while they could also react to chains of DP5-10 and 7-13 to some extent, respectively. In contrast, all these ISAs were reactive to various chains when incubated with amylopectin. In addition to a great variation in chain-length preferences among various ISAs, their activities greatly differed depending on a variety of glucans. Most strikingly, cyannobacteria ISAs could attack branch points of pullulan to a lesser extent although no such activity was found in OsISA1, OsISA3, EcoGlgX, and PsaISA. Thus, the present study shows the high possibility that varied chain-length specificities of ISA-type DBEs among sources and isozymes are responsible for their distinct functions in glucan metabolism.
Assuntos
Bactérias/genética , Cianobactérias/genética , Sistema da Enzima Desramificadora do Glicogênio/química , Isoamilase/química , Oryza/enzimologia , Bactérias/enzimologia , Cianobactérias/enzimologia , Endosperma/enzimologia , Glucanos/química , Glucanos/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Isoamilase/genética , Oryza/genética , Amido/químicaRESUMO
The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light-dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.
Assuntos
Proteínas de Bactérias/metabolismo , Genes Bacterianos , Glicogênio/metabolismo , Synechococcus/genética , Synechococcus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Cromossomos Bacterianos , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/genética , Glicogênio/química , Glicogênio/genética , Isoamilase/metabolismo , Luz , Dados de Sequência Molecular , Mutação , Consumo de Oxigênio , Fotossíntese , Mapeamento Físico do Cromossomo , Homologia de Sequência de Aminoácidos , Synechococcus/enzimologiaRESUMO
Eimeria necatrix-specific ELISA, using a recombinant antigen (the cDNA-clone NP19 expressing protein), was utilized to detect antibodies against E. necatrix in breeder pullet flocks that had previously received an attenuated live vaccine to E. necatrix. Vaccinated flocks were discriminated significantly from non-vaccinated flocks by their antibody titers and antibody positive rates at 30-55 days post-vaccination. In addition, E. necatrix-oocysts were confirmed in fecal samples of vaccinated flocks using PCR in the case where the antibody positive rates rose. These findings implied that the vaccination prompted repeated infections, and consequently the chickens generated antibodies and secured their protection against virulent field-E. necatrix. Therefore, the ELISA was suggested to be a useful tool to estimate the immune state of chickens as a result of vaccination with a live E. necatrix-vaccine.
