scholarly journals Galactose transfer to endogenous acceptors within Golgi fractions of rat liver.

1982 ◽  
Vol 92 (1) ◽  
pp. 139-146 ◽  
Author(s):  
J J Bergeron ◽  
R A Rachubinski ◽  
R A Sikstrom ◽  
B I Posner ◽  
J Paiement
Keyword(s):  
Rat Liver ◽  
Transferase Activity ◽  
Cis Elements ◽  
Galactosyl Transferase ◽  
Highly Active ◽  
Cell Biol ◽  
Step Procedure ◽  
One Step ◽  
Low Activity

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.

scholarly journals Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

1977 ◽  
Vol 165 (1) ◽  
pp. 121-126 ◽  
Author(s):  
G D Smith ◽  
D V Roberts ◽  
A Daday
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibitor ◽  
Chorismate Mutase ◽  
Prephenate Dehydrogenase ◽  
Step Procedure ◽  
One Step ◽  
High Degree ◽  
Sepharose 4B ◽  
Derivatives Of

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1971 ◽  
Vol 49 (3) ◽  
pp. 899-905 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Carol Pannek ◽  
Daniel S. Friend
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Total Activity ◽  
Thiamine Pyrophosphate ◽  
Transferase Activity ◽  
Galactosyl Transferase ◽  
Thiamine Pyrophosphatase ◽  
Cell Components

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

scholarly journals Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

1977 ◽  
Vol 164 (3) ◽  
pp. 685-691 ◽  
Author(s):  
E Marra ◽  
S Doonan ◽  
C Saccone ◽  
E Quagliariello
Keyword(s):  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Transferase Activity ◽  
Selective Permeability ◽  
The Matrix ◽  
Mitochondrial Aspartate Aminotransferase

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

scholarly journals Precocious development of uridine diphosphate glucuronosyltransferase activity during organ culture of foetal rat liver in the presence of glucocorticoids

1977 ◽  
Vol 166 (2) ◽  
pp. 249-253 ◽  
Author(s):  
G J Wishart ◽  
M A Goheer ◽  
J E A Leakey ◽  
G J Dutton
Keyword(s):  
Rat Liver ◽  
Defined Medium ◽  
Transferase Activity ◽  
Uridine Diphosphate ◽  
Chemically Defined Medium ◽  
Foetal Rat ◽  
First Time ◽  
Precocious Development ◽  
Stimulation Of

1. Precocious development of mammalian UDP-glucuronosyltransferase (EC 2.4.1.1.7) induced by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs in cultured explants of foetal rat liver when exposed to corticosteroids possessing a pregn-4′-ene structure and a hydroxy or an oxo group at C-11. 3. Explants from 14-day foetuses cultured for 3 days in a chemically defined medium containing dexamethasone exhibited transferase activities towards o-aminophenol within adult male values. Those liver transferase activities attained in utero by 17 days were still negligible. 4. Evidence from several approaches indicated that the explants required glucocorticoids for expression of the transferase, not for maintenance of viability. 5. Glucocorticoid-dependent stimulation of transferase activity required incorporation of L-[14C]leucine into protein, as judged from the pulsing of cultures with cycloheximide. 6. The relevance of these culture experiments to the situation in vivo is discussed.

scholarly journals Fusogenic Domains of Golgi Membranes Are Sequestered into Specialized Regions of the Stack that Can Be Released by Mechanical Fragmentation

1999 ◽  
Vol 145 (4) ◽  
pp. 673-688 ◽  
Author(s):  
Michel Dominguez ◽  
Ali Fazel ◽  
Sophie Dahan ◽  
Jacque Lovell ◽  
Louis Hermo ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Maximal Activity ◽  
Anterograde Transport ◽  
Galactosyl Transferase ◽  
Highly Active ◽  
Golgi Transport ◽  
Transport Assay ◽  
Mechanical Fragmentation

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.

scholarly journals Expansion of Submucosal Bladder Wall TissueIn VitroandIn Vivo

2016 ◽  
Vol 2016 ◽  
pp. 1-9
Author(s):  
Gisela Reinfeldt Engberg ◽  
Clara Ibel Chamorro ◽  
Agneta Nordenskjöld ◽  
Magdalena Fossum
Keyword(s):  
Smooth Muscle ◽  
Bladder Wall ◽  
Subcutaneous Fat ◽  
Culture Conditions ◽  
Detrusor Muscle ◽  
Step Procedure ◽  
Standard Culture ◽  
One Step

In order to develop autologous tissue engineering of the whole wall in the urinary excretory system, we studied the regenerative capacity of the muscular bladder wall. Smooth muscle cell expansion on minced detrusor musclein vitroandin vivowith or without urothelial tissue was studied. Porcine minced detrusor muscle and urothelium were culturedin vitrounder standard culture conditions for evaluation of the explant technique and in collagen for tissue sectioning and histology. Autografts of minced detrusor muscle with or without minced urothelium were expanded on 3D cylinder moulds by grafting into the subcutaneous fat of the pig abdominal wall. Moulds without autografts were used as controls. Tissue harvesting, mincing, and transplantation were performed as a one-step procedure. Cells from minced detrusor muscle specimens migrated and expandedin vitroon culture plastic and in collagen.In vivostudies with minced detrusor autografts demonstrated expansion and regeneration in all specimens. Minced urothelium autografts showed multilayered transitional urothelium when transplanted alone but not in cotransplantation with detrusor muscle; thus, minced bladder mucosa was not favored by cografting with minced detrusor. No regeneration of smooth muscle or epithelium was seen in controls.

scholarly journals Biogenesis of peroxisomes: isolation and characterization of two distinct peroxisomal populations from normal and regenerating rat liver.

1993 ◽  
Vol 121 (6) ◽  
pp. 1271-1280 ◽  
Author(s):  
G Lüers ◽  
T Hashimoto ◽  
H D Fahimi ◽  
A Völkl
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Form Factors ◽  
Biochemical Properties ◽  
Peroxisomal Membrane ◽  
Homogeneous Population ◽  
Isolation And Characterization ◽  
Cell Biol

According to Poole et al. (1970, J. Cell Biol. 45:408-415), newly synthesized peroxisomal proteins are incorporated uniformly into peroxisomes (PO) of different size classes, suggesting that rat hepatic PO form a homogeneous population. There is however increasing cytochemical and biochemical evidence that PO in rat liver are heterogenous, undergoing significant modulations in shape and size in process of PO morphogenesis (Yamamoto and Fahimi, 1987. J. Cell Biol. 105:713-722). In the present study, the kinetics of incorporation of newly synthesized proteins into distinct PO-subpopulations have been studied using short-term in vivo labeling (5-90 min). Two distinct "heavy" and "light" crude PO fractions were prepared by differential pelleting from normal and regenerating liver, and highly purified PO were subsequently isolated by density-dependent metrizamide gradient centrifugation according to Völkl and Fahimi (1985. Eur. J. Biochem. 149:257-265). The peroxisomal fractions banded at 1.20 and 1.24 g x cm-3. They differed in their mean diameters and form-factors and particularly in respect to the activity of beta-oxidation enzymes which was higher in the "light" PO. Whereas the "light" PO exhibited a single immunoreactive band with the antibody to the 70-kD peroxisomal membrane protein the "heavy" PO contained an additional (68 kD) band. In pulse-labeling experiments "light" PO showed clearly a higher initial rate of incorporation than the "heavy" PO. The relative specific activity in the "heavy" PO fraction, however increased progressively reaching that of "light" PO by 90 min. These observations provide evidence for the existence of different PO populations in rat liver which differ in their morphological and biochemical properties as well as in their rates of incorporation of new proteins.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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