Calcium Enhances The Proteolytic Activity Of BACE: An In Vitro Biophysical And Biochemical Characterization Of The BACE-Calcium Interaction

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.427 ◽

1985 ◽

Vol 101 (2) ◽

pp. 427-440 ◽

Cited By ~ 40

Author(s):

E Bartnik ◽

M Osborn ◽

K Weber

Keyword(s):

Positive Reaction ◽

Intermediate Filaments ◽

Biochemical Characterization ◽

Helix Pomatia ◽

Molar Ratio ◽

Negative Stain ◽

Epithelial Morphology ◽

Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

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Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

The Journal of Cell Biology ◽

10.1083/jcb.93.1.144 ◽

1982 ◽

Vol 93 (1) ◽

pp. 144-154 ◽

Cited By ~ 106

Author(s):

L Marzella ◽

J Ahlberg ◽

H Glaumann

Keyword(s):

Proteolytic Activity ◽

Rat Liver ◽

Biochemical Characterization ◽

Secretory Granules ◽

Density Lipoprotein ◽

Marker Enzymes ◽

Cell Organelles ◽

Protein Ratio ◽

Autophagic Vacuoles

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

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In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

Methods in Molecular Biology - Yeast Cytokinesis ◽

10.1007/978-1-4939-3145-3_12 ◽

2016 ◽

pp. 151-179 ◽

Cited By ~ 11

Author(s):

Dennis Zimmermann ◽

Alisha N. Morganthaler ◽

David R. Kovar ◽

Cristian Suarez

Keyword(s):

Binding Proteins ◽

Biochemical Characterization ◽

Actin Binding ◽

Actin Binding Proteins

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Biochemical characterization of HIV-1 Rev as a potent activator of casein kinase II in vitro

FEBS Letters ◽

10.1016/s0014-5793(98)00538-9 ◽

1998 ◽

Vol 428 (3) ◽

pp. 235-240 ◽

Cited By ~ 26

Author(s):

Kenzo Ohtsuki ◽

Toshiro Maekawa ◽

Shigeyoshi Harada ◽

Atsushi Karino ◽

Yuko Morikawa ◽

...

Keyword(s):

Biochemical Characterization ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Hiv 1

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Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

The Journal of Biochemistry ◽

10.1093/jb/mvz010 ◽

2019 ◽

Vol 166 (1) ◽

pp. 51-66 ◽

Cited By ~ 2

Author(s):

Jan M Klenk ◽

Max-Philipp Fischer ◽

Paulina Dubiel ◽

Mahima Sharma ◽

Benjamin Rowlinson ◽

...

Keyword(s):

Cytochrome P450 ◽

Metabolic Pathway ◽

Biochemical Characterization ◽

Gene Clusters ◽

Cytochrome P450 Monooxygenases ◽

Dihydroxyphenylacetic Acid ◽

Meta Position ◽

Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

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