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.     

An ultrastructural study of centriolar complexes in adult and embryonic human aortic endothelial cells

Ultrastructural organization of centriolar complexes in 90 adult human aortic endothelial cells from uninvolved areas, fibrous and atheromatous plaques and 30 endothelial cells from human embryonic aorta were studied using serial sections. Primary cilia protruding from the abluminal cell surface were found on 28 of 30 endothelial cells from atheromatous plaques. Only five of 30 cells from either fibrous plaques or uninvolved areas developed primary cilia protruding to the lumen. Impaired primary cilia entirely immersed into the cytoplasm were found in embryonic endothelial cells. It was speculated that both the modes of formation and the functions of endothelial cilia in embryonic and adult aortas are different.     

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.     

Ca2+ ions induce successive capacity and ion currents in BLM from phosphatidic acid as a result of phase transition

Time dependence of Ca2+-induced electric current in BLM formed from DPPA was studied at constant temperature and pH. The phase transition in BLM is accompanied by capacity current and following appearance of single ionic channels. It was shown that transferred charge was 5 nC/F, conductivity of single ionic channels--500-100 pSm.     

Study of the structural and functional properties of fibronectin--a high molecular weight plasma glycoproteins--by using monoclonal antibodies

Functional and structural properties of fibronectin--high molecular weight glyco-protein from human plasma--were studied by monoclonal antibodies against fibronectin. It was shown that monoclonal antibodies against human plasma fibronectin exhibit a certain species specificity. Antigenic determinant for our monoclonal antibody is located in the central part of the protein polypeptide chain--in the structural domain. The monoclonal antibodies studied do not inhibit any tested functions of fibronectin. In contrast, polyclonal antibodies are not species specific and inhibit all fibronectin functions.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

“Maturation” of DNA duplexes

Reassociation of typical single-copy DNAs, like E. coli DNA, even when performed at relatively low temperatures, results in the formation of perfect duplexes with thermal stability very close to that of the native DNA. In contrast, duplexes of mouse repeated DNA as well as duplexes of Streptomyces DNA prepared under the same conditions, show a low thermal stability and undergo post-reassociation changes upon prolonged incubation. These changes, called maturation of the DNA duplexes, result in increasing of their thermal stability. Some of the factors affecting the rate of maturation are studied. The implication of the maturation process in reassociation analysis and in characterization of the heterogeneity of DNA is discussed.     

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.