Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium

1989 ◽  
Vol 14 (7) ◽  
pp. 689-691 ◽  
Author(s):  
Anna Baranczyk-Kuzma ◽  
Kenneth L. Audus ◽  
Ronald T. Borchardt
Keyword(s):  
Substrate Specificity ◽  
Primary Cultures ◽  
Bovine Brain ◽  
Phenol Sulfotransferase ◽  
Brain Microvessel

Hexose uptake in primary cultures of bovine brain microvessel endothetial cells. I. Basic characteristics and effects of d-glucose and insulin

1991 ◽  
Vol 1070 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Yoshinobu Takakura ◽  
Sandra L. Kuentzel ◽  
Thomas J. Raub ◽  
Anthony Davies ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Basic Characteristics ◽  
Hexose Uptake ◽  
Brain Microvessel

scholarly journals Demonstration of Acid Hydrolase Activity in Primary Cultures of Bovine Brain Microvessel Endothelium

1989 ◽  
Vol 9 (3) ◽  
pp. 280-289 ◽  
Author(s):  
Anna Baranczyk-Kuzma ◽  
Thomas J. Raub ◽  
Kenneth L. Audus
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Endocytic Pathway ◽  
Hydrolase Activity ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Cytochemical Localization ◽  
Brain Microvessel

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood–brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and β-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69–77%). The majority of β-galactosidase (≈48%) and total sulfatase (≈58%) activity was associated with the lysosome fraction of the BMECs. In contrast, ≈52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

scholarly journals Characteristics of Aminopeptidase Activity from Bovine Brain Microvessel Endothelium

1987 ◽  
Vol 7 (6) ◽  
pp. 801-805 ◽  
Author(s):  
Anna Baranczyk-Kuzma ◽  
Kenneth L. Audus
Keyword(s):  
Competitive Inhibition ◽  
Primary Cultures ◽  
Bovine Brain ◽  
Aminopeptidase Activity ◽  
Membrane Bound ◽  
Vitro Model ◽  
Brain Microvessel ◽  
Aminopeptidase Inhibitors ◽  
Assay Conditions

Blood–brain barrier (BBB) aminopeptidase activity was investigated using an in vitro model consisting of primary cultures of brain microvessel endothelium. Using two different substrates, both membrane-bound and soluble aminopeptidases were found to be associated with brain endothelium. That the enzyme activity was aminopeptidase activity was confirmed with the competitive inhibition of substrate degradation by typical aminopeptidase inhibitors puromycin and bestatin. The aminopeptidase activity was also competitively inhibited by enkephalin, met-enkephalin, and leu-enkephalin. Results from parallel experiments with cerebral gray matter and kidney confirm assay conditions. This report supports previous suggestions that aminopeptidases of the enzymatic BBB may play a role in regulating levels of circulating neuropeptides in the cerebrovasculature.

scholarly journals Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

1990 ◽  
Vol 97 (1) ◽  
pp. 127-138
Author(s):  
T.J. Raub ◽  
K.L. Audus
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Basolateral Membrane ◽  
Surface Membrane ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Bovine Brain ◽  
Membrane Recycling ◽  
Brain Microvessel

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13–14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

1999 ◽  
Vol 73 (5) ◽  
pp. 3737-3743 ◽  
Author(s):  
Alfred Klausegger ◽  
Birgit Strobl ◽  
Gerhard Regl ◽  
Alexandra Kaser ◽  
Willem Luytjes ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Solid Phase ◽  
Hepatitis Virus ◽  
Bovine Brain ◽  
Brain Extract ◽  
Binding Assays ◽  
Bovine Coronavirus ◽  
Close Relationship ◽  
Influenza C Virus ◽  
Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Primary cultures from fetal bovine brain

Neuroreport ◽  
2004 ◽  
Vol 15 (11) ◽  
pp. 1719-1722 ◽  
Author(s):  
Antonella Peruffo ◽  
Maria Lina Massimino ◽  
Cristina Ballarin ◽  
Giorgio Carmignoto ◽  
Ada Rota ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Fetal Bovine
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