Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Detergent properties of apolipoprotein A1 in micellar complexes with dimyristoyl phosphatidylcholine

Bilayer micellar complexes of the human plasma apolipoprotein A1 with dimyristoyl phosphatidylcholine were prepared under kinetically controlled conditions. Detergent-like properties of Apo A1 in the complexes were expressed in terms of delta SA1 parameter (surface area of mixed micelle per an Apo A1 molecule). Analysis of our and earlier published data showed the correlation of the delta SA1 parameter with the stoichiometry of complexes. Changes of detergent-like properties were caused by cross-linking or proteolysis of Apo A1.     

The structure of the centriolar apparatus of the endothelial cells in the embryonic and definitive human aorta

The fine structure of the centriolar system was studied on serial sections of 90 endothelial cells of human aorta (50 to 60 years) in regions without atherosclerotic platelets and with fibrous and atheromatous platelets and of 30 endothelial cells of human embryonic aorta (22-24 weeks). The vast majority (95%) of endothelial cells of the atheromatous platelets were shown to have a primary cilium over 1 micron long which gives on the basal surface in all the cells. In the regions without platelets and with fibrous platelets a cilium was observed in about 20% of cells and it gives in the vessel lumen. Endothelial cells with a cilium fully embedded in the cytoplasm and with abnormal cilium structure were found in the embryonic aorta. A suggestion is put forward that cilia of the endothelial cells of embryonic aorta and those of adult aorta differ by the mechanism of their formation and can have different functions.     

Aldosterone and electrolyte content in blood and myocardium of rats after single physical load in acute alcoholic intoxication]

It is shown that acute alcoholic intoxication causes essential changes of aldosterone content and balance of electrolytes in blood plasma of Wistar rat males. The SPL test revealed the inadequate reaction in the mineral-corticoid function of the adrenal glands in the rats with acute alcoholic intoxication. The method of the free choice of the load by the experimental rats is the most optimal way to assess. The rats with acute alcoholic intoxication displayed smaller tolerance to physical load.     

Determination of leucine-binding protein content of Escherichia coli cells]

A radioimmunochemical method of determination of leucine-binding protein and a method, based on the selective absorption of the protein and its complex with leucine on DEAE-cellulose, has been developed. The protein content in the E. coli cells at different stage of growth has been determined by the radioimmunochemical and equilibrium dialysis methods. It was shown that the protein content in the cells is practically independent of the growth-phase.     

Electrophoresis of chloride ions and changes in the membrane potential of energized mitochondria]

In energized rat liver mitochondria the simultaneous H+, K+ and C1- transport was studied by corresponding ion selective electrodes. It was shown that the C1- transport induced by valinomycin, by valinomycin plus carbonyclyanide m-chlorophenylhydrazone was governed by the membrane potential. It is suggested that observed in energized mitochondria the C1- electrophoresis may servt as an indicator of membrane potential changes.