mersalyl and leupeptin

Topics: Adenosine; Allopurinol; Animals; Benzamidines; Creatinine; Dogs; Female; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Leupeptins; Mersalyl; Mitochondria; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Perfusion; Quinacrine; Raffinose; Solutions; Time Factors

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.

Topics: Anion Exchange Protein 1, Erythrocyte; Blood Proteins; Calcimycin; Calpain; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Leupeptins; Membrane Proteins; Mersalyl; Neuropeptides; Spectrin

The mechanisms controlling the reorganisation of synaptic inputs to developing skeletal muscle fibres was studied using electrophysiological and histological methods. In the developing rat soleus muscle there is a rapid reduction of polyneuronal innervation between 9 and 12 days. Reducing the local concentration of calcium by applying chelating agents such as EGTA or BAPTA in vivo to 9-day-old rat soleus muscles over a period of 3 days slowed the rate of elimination of polyneuronal innervation. It was established that the reduction of calcium induced by EGTA or BAPTA was not sufficient to produce a detectable reduction in neuromuscular activity. The possibility that a calcium-dependent enzyme such as CANP may play a role in synapse reorganisation was therefore tested. Local application of inhibitors of calcium-activated neutral protease (CANP), leupeptin or E-64, to 9-day-old rat soleus muscles over 3 days had similar effects to those of EGTA or BAPTA, i.e. the elimination of polyneuronal innervation that usually takes place was much slower. Since the inhibition of thiol proteases had similar effects on synapse elimination as a reduction of calcium concentration, it is concluded that CANP is important in the reorganisation of the developing neuromuscular junction.

Topics: Animals; Animals, Newborn; Calcium; Calpain; Egtazic Acid; Leupeptins; Mersalyl; Muscle Development; Neuromuscular Junction; Rats; Rats, Inbred Strains

Mechanisms controlling the local generation of angiotensin II by vascular tissue are incompletely understood. Human platelets were examined for their ability to metabolize angiotensin I. Platelet-dependent angiotensin I metabolism was detected by a high performance liquid chromatography assay which allowed quantitation of angiotensin I substrate utilized and products formed. The major product of platelet-dependent angiotensin I metabolism was identified as des-Leu10-angiotensin I. The platelet des-Leu10-angiotensin I-generating activity had a pH optimum of 6.0-6.5 and was inhibited 100% by mersalyl acid (10(-4) M), 86% by leupeptin (10(-4) M), and 95% by iodoacetamide (10(-2) M). The activity had an approximate Mr = 70,000 as determined by Sephacryl S-200 gel filtration. Intact human platelets stimulated with calcium ionophore (1-10 microM) released 13.7-30.8% of the des-Leu10-angiotensin I-generating activity. Des-Leu10-angiotensin I, the major product of platelet angiotensin I metabolism, inhibited human serum and purified rabbit lung angiotensin-converting enzymes with an I50 of 3.7 X 10(-6) and 2.0 X 10(-6) M, respectively. These results suggest that the platelet may control local angiotensin II formation at vascular sites both by metabolism of the precursor peptide angiotensin I and by generation of an endogenous angiotensin-converting enzyme inhibitor, des-Leu10-angiotensin I. This platelet-dependent pathway may contribute to the control of local levels of vasoactive peptides, such as bradykinin and angiotensin II, so as to alter local tissue blood flow.

Topics: Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Angiotensins; Animals; Blood Platelets; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Hydrogen-Ion Concentration; Iodoacetamide; Leupeptins; Lung; Mersalyl; Molecular Weight; Rabbits