INCREASE IN THE APPARENT AChE ACTIVITY IN THE MOUSE DIAPHRAGM INDUCED BY PROTEOLYTIC TREATMENT

Abstract— Isolated endplate regions from the mouse diaphragm were treated with different agents before or after homogenization in order to solubilize junctional AChE and study the effect of solubilization on its apparent activity. Total AChE activity (solubilized + nonsolubilized) of samples treated with collagenase or papain before homogenization was nearly twice as high as in control samples. If collagenase was added after homogenization no increase in apparent activity was observed although in both cases about 70–80% of AChE activity was solubilized. The access of ACh to the membrane-bound enzyme is probably not a limiting factor in the AChE assay as is the case in the electric organ homogenates. Both 1 m -NaCl and Triton X-100 were quite ineffective as solubilizers when applied before homogenization and had an insignificant effect on the apparent AChE activity.
The increase in apparent AChE activity cannot be explained either by a de novo synthesis or by the change in kinetic properties of different species of AChE, or by the release of AChE possibly sequestrated in the membrane vesicles. The possibility is discussed that a part of junctional AChE is inactivated at the beginning of homogenization while it can be preserved by previous solubilization, or that proteolytic treatment may activate some 'silent' AChE sites in motor endplates.
However, the mere fact that the difference does exist suggests that all AChE activity present in intact motor endplates may not be measurable after homogenization.     

The binding of IMP to ribonuclease A

The binding of inosine 5' phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray crystallographic experiments at high (1.5 A) resolution. IMP is a competitive inhibitor of the enzyme with respect to C>p and binds to the catalytic cleft by anchoring three IMP molecules in a novel binding mode. The three IMP molecules are connected to each other by hydrogen bond and van der Waals interactions and collectively occupy the B1R1P1B2P0P(-1) region of the ribonucleolytic active site. One of the IMP molecules binds with its nucleobase in the outskirts of the B2 subsite and interacts with Glu111 while its phosphoryl group binds in P1. Another IMP molecule binds by following the retro-binding mode previously observed only for guanosines with its nucleobase at B1 and the phosphoryl group in P(-1). The third IMP molecule binds in a novel mode towards the C-terminus. The RNase A-IMP complex provides structural evidence for the functional components of subsite P(-1) while it further supports the role inferred by other studies to Asn71 as the primary structural determinant for the adenine specificity of the B2 subsite. Comparative structural analysis of the IMP and AMP complexes highlights key aspects of the specificity of the base binding subsites of RNase A and provides a structural explanation for their potencies. The binding of IMP suggests ways to develop more potent inhibitors of the pancreatic RNase superfamily using this nucleotide as the starting point.     

Localization and activity of cholinesterases in Bidder's ganglion cells and nerve fibers of the frog heart

Summary The histochemical and cytochemical localization of cholinesterases in Bidder's ganglion of the frog heart was investigated and the activity of cholinesterases measured microgasometrically.When either acetylthiocholine or propionylthiocholine was used as substrate, the end product of histochemical and cytochemical reaction was found in all ganglion cells and nerve fibers, the only difference being in the amount of the accumulated precipitate. The end product was localized on the axolemma and in the smooth endoplasmic reticulum of nerve fibers; in the smooth and granulated endoplasmic reticulum of ganglion cells; in the synaptic region between the nerve ending and Schwann cells membrane; and, occasionally, in discontinous streches between the pre- and postsynaptic membrane. The nerve endings of the neurons of the Bidder's ganglion contain predominantly agranular vesicles with occasional clusters of granular ones.Neither the histochemical nor the cytochemical localization of enzyme activity could be detected when butyrylthiocholine was used as substrate. In microgasometric experiments splitting of butyrylcholine was hardly, if at all, measurable. Cholinesterases of the ganglion cells and of the nerve fibers of Bidder's ganglion hydrolyze propionylcholine but at a somewhat lower rate than acetylcholine.A part of this work has been presented at the VI. Congress of the Yugoslav Physiological Society, Ohrid, 1969.This work was supported by the Boris Kidri Foundation, Ljubljana, and by the Federal Research Council, Beograd.     

A comparison between the one-step and the two-step copper thiocholine procedure for the cytochemical localization of cholinesterases

Summary A comparison between the one-step and the two-step copper thiocholine procedure for the subcellular localization of cholinesterases was under-taken. The results indicate that only under experimental conditions with short incubation times and precise control of the conversion into sulphide is the localization of the primary precipitate and that of the secondary precipitate identical. It was concluded that the conversion of the primary precipitate into Cu-sulphide is not necessary and can lead to artefacts.     

Influence of UV-B exclusion and selenium treatment on photochemical efficiency of photosystem II, yield and respiratory potential in pumpkins (Cucurbita pepo L.).

The influence of ambient and filtered solar ultraviolet-B (UV)-B radiation and of selenium treatment was determined on photochemical efficiency, respiratory potential measured by electron transport system (ETS) activity, and yield in pumpkins, Cucurbita pepo L. Yield and ETS activity were higher when solar UV-B was filtered out. The results suggested that the decreased yield was related to the UV-B impaired flow of electrons in the respiratory chain. Selenium increased yield under ambient radiation conditions. However, no significant effect of excluding UV-B radiation or of treatment with selenium was observed on the photochemical efficiency of photosystem II (PSII).     

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme

Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1-6), dynorphin (1-13), alpha-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid D-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1-13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1-6), alpha-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [delta1-10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10(-10) M range.     

A novel subtilase from common bean leaves

We describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.6, and SDS–PAGE in a single band corresponding to M_r 72 kDa. The proteinase activity is maximal at pH 9.9 and shows high stability in the alkaline region. The relative activities of the proteinase for eight different synthetic substrates were determined. The requirement for Arg in the P1 position appeared obligatory. k_cat/K_m values indicate that, for highest catalytic efficiency, a basic amino acid is also required in the P2 position, presenting a motif typical of the cleavage site for the kexin family of subtilases. The sequence of the 17 N-terminal amino acids of this proteinase shows similarity to those of other plant subtilases, sharing the highest number of identical amino acids with proteinase C1 from soybean seedling cotyledons and a cucumisin-like proteinase from white gourd (Benincasa hispida).     

CHOLINESTERASE ACTIVITY OF NODAL AND INTERNODAL REGIONS OF MYELINATED NERVE FIBERS OF FROG

The distribution of cholinesterase (Ch-esterase) in isolated myelinated fibers of the frog has been investigated. Quantitative microgasometric measurements have confirmed the previous histochemical observations. Both approaches indicate that in frog nerve fibers acetylcholinesterase (ACh-esterase) is the only or the predominant enzyme when selective inhibitors and different substrates are used: acetylcholine (ACh), butyrylcholine, and acetyl-B-methylcholine (Mecholyl). By means of the microgasometric technique, a significant difference in ACh-esterase activity between axons isolated from ventral (37.2 ± 1.7 µmole x 10^-5 ACh/mm²/hr) and dorsal roots (2.0 ± 0.9 µmole x 10^-5 ACh/mm²/hr) was found. In the region of the node of Ranvier the enzyme activity (50.4 ± 4.4 µmole x 10^-5 ACh/mm²/hr) appears to be considerably higher than in the internodal area (36.6 ± 2.1 µmole x 10^-5 ACh/mm²/hr). The findings are discussed in relation to the theory of saltatory conduction and the ACh system.