The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

A new synaptic anomaly: irregular synaptonemal complexes

Summary In this paper we describe a new synaptic anomaly characterized by the presence of irregular synaptonemal complexes (SCs) in two sterile patients.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Interaction of concanavalin A with sugar residues of collagen from different tissues

Topochemical characteristics of reactions of different types of collagen-containing structures with Concanavalin A (Con A) have not been considered up to now. In this study the presence and availability of glucose residues of collagen molecules from intestine, liver, cartilage and tendon are detected using Con A and horseradish peroxidase (HRP). In intestine, cartilage and tendon sections, the Con A-HRP method was only significantly positive when the sections were first submitted to treatment with papain. This suggested the presence of glycoproteins and proteoglycans of the extracellular matrix (ECM), which might interfere either interacting with lateral sugar residues of the collagen molecules, or causing some steric blockade or even masking as occurs in regions with a high state of compactness.     

A murine sequence-specific DNA binding protein shows extensive local similarities to the amyloid precursor protein.

Microinjection experiments suggested previously that protein binding to the DNA nucleotide sequence GTCACATG, identical to the CDEI element of the yeast centromere, plays an important role in the early development of the mouse. We established from a series of overlapping mouse cDNA clones the sequence of a candidate CDEI-binding protein. Synthesis in Escherichia coli of a fusion protein which binds specifically the CDEI box in vitro confirmed its identification. On the other hand, the translated 511 amino acid sequence shows two regions with high degrees of similarity to the protein precursor (APP) of the beta-protein (amyloid) that accumulates in the brain and blood vessels of Alzheimer patients. A continuous stretch of 195 amino acids includes 133 residues identical to part of the extracellular domain of APP, and 48 of the 70 C-terminal residues of the open reading frame are identical to the APP transmembrane and cytoplasmic domains.     

A stuttering model for paramyxovirus P mRNA editing.

Paramyxovirus P genes are transcribed into two mRNAs which differ from each other by either one (measles and Sendai virus) or two (SV5 and mumps virus) G insertions, and which code for either the P or V proteins. The G insertions always occur within a short run of Gs, and a stuttering mechanism for the insertions has been suggested in which the viral polymerase reiteratively copies a template C residue during mRNA synthesis. Support for this mechanism was obtained by varying the reaction conditions during Sendai virus mRNA synthesis in vitro. A stuttering model is proposed which accounts for how the ratio of inserted to uninserted mRNAs is controlled, and why some paramyxoviruses insert one G and others two Gs when insertions occur.     

Enhancer sequences located 3' of the mouse immunoglobulin lambda locus specify high-level expression of an immunoglobulin lambda gene in B cells of transgenic mice

In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system.