THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3).

The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.     

Molecular cloning of rabbit CARP cDNA and its regulated expression in adriamycin-cardiomyopathy.

A full-length rabbit cDNA of cardiac adriamycin responsive protein (CARP) has been cloned. It shows high levels of identity at the amino acid sequence level (>86%) with the rat, mouse and human homologues. CARP mRNA levels are highly regulated in adriamycin-cardiomyopathy in rabbits.     

Integrating the statistical analysis of spatial data in ecology

In many areas of ecology there is an increasing emphasis on spatial relationships. Often ecologists are interested in new ways of analyzing data with the objective of quantifying spatial patterns, and in designing surveys and experiments in light of the recognition that there may be underlying spatial pattern in biotic responses. In doing so, ecologists have adopted a number of widely different techniques and approaches derived from different schools of thought, and from other scientific disciplines. While the adaptation of a diverse array of statistical approaches and methodologies for the analysis of spatial data has yielded considerable insight into various ecological problems, this diversity of approaches has sometimes impeded communication and retarded more rapid progress in this emergent area. Many of these different statistical methods provide similar information about spatial characteristics, but the differences among these methods make it difficult to compare the results of studies that employ contrasting approaches. The papers in this mini-series explore possible areas of agreement and synthesis between a diversity of approaches to spatial analysis in ecology.     

Correlation between the bone mass of athletes and biochemical and genetic markers of bone tissue remodeling

The association of the polymorphism of the VDR, Col1a1, and CALCR genes with a form of osteoporosis frequently occurring as a consequence of intense physical exercise in athletes was studied. Biochemical parameters of bone remodeling and its neuroendocrine regulation, as well as the bone masses, of 22 amateur athletes were determined immediately before a strenuous nine-week training cycle (TC) and eight months later. The possible association of these factors with the polymorphism of the genes coding for bone tissue proteins was studied. Long-term intense physical training was found to be associated with a significant activation of bone tissue resorption accompanied by continued rapid synthesis. Nevertheless, and in spite of the strong activation of resorption caused by the TC, the athletes exhibited no osteoporosis (even eight months after the discontinuation of the TC); some of them, however, displayed an individual tendency to osteopenia. According to the results of genetic analysis, this was associated with the polymorphism of predisposition genes (genotype TT of the VDR gene and the functionally weakened s allele of the Col1a1 gene).