scholarly journals Enzyme activity after resealing within ghost erythrocyte cells, and protection by α-crystallin against fructose-induced inactivation

2002 ◽  
Vol 368 (3) ◽  
pp. 865-874 ◽  
Author(s):  
Barry K. DERHAM ◽  
John J. HARDING
Keyword(s):  
Enzyme Activity ◽  
Molecular Chaperone ◽  
Soluble Fraction ◽  
Erythrocyte Ghosts ◽  
Ghost Cell ◽  
Chaperone Function ◽  
Zero Time ◽  
Soluble Fractions

The role of α-crystallin as a molecular chaperone has been shown in many in vitro studies. In the present paper, we report on the chaperone function of α-crystallin within resealed erythrocyte ghosts. Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24h. The ghost cell suspension was separated into soluble and membrane fractions. Five of the enzymes had significantly greater enzyme activity after 24h than the control within the soluble fractions. Fructation caused a decrease in enzyme activity (relative to the control). Resealing of α-crystallin within the ghost cell alongside the enzymes protected against inactivation by fructose within the soluble fraction.

scholarly journals Effects of modifications of α-crystallin on its chaperone and other properties

2002 ◽  
Vol 364 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Barry K. DERHAM ◽  
John J. HARDING
Keyword(s):  
Congenital Cataract ◽  
Point Mutations ◽  
Small Heat Shock Protein ◽  
High Molecular Mass ◽  
Lens Crystallins ◽  
Post Translational Modifications ◽  
Chaperone Function ◽  
Arginine Residues

The role of α-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. α-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by ‘trapping’ the protein in a high-molecular-mass complex. However, during aging, the chaperone function of α-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts. Within the central part of the lens there is no turnover of damaged protein, and therefore post-translational modifications of α-crystallin accumulate that can reduce chaperone function; this is compounded in cataract lenses. Extensive in vitro glycation, carbamylation and oxidation all decrease chaperone ability. In the present study, we report the effect of the modifiers malondialdehyde, acetaldehyde and methylglyoxal, all of which are pertinent to cataract. Also modification by aspirin, which is known to delay cataract and other diseases, has been investigated. Recently, two point mutations of arginine residues were shown to cause congenital cataract. 1,2-Cyclohexanedione modifies arginine residues, and the extent of modification needed for a change in chaperone function was investigated. Only methylglyoxal and extensive modification by 1,2-cyclohexanedione caused a decrease in chaperone function. This highlights the robust nature of α-crystallin.

Calcium ATPase and intestinal calcium transport in uremic rats

1980 ◽  
Vol 238 (5) ◽  
pp. G424-G428
Author(s):  
H. Schiffl ◽  
U. Binswanger
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Calcium Transport ◽  
Calcium Atpase ◽  
Intestinal Calcium Transport ◽  
Identical Response ◽  
Close Relationship ◽  
Intact Rats

Calcium ATPase, an enzyme involved in intestinal calcium transport, was measured in homogenates of duodenal mucosal scrapings of normal and uremic rats. The effects of calcium deprivation and treatment with 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3] were investigated as well. Uremia decreased the enzyme activity and impaired the rise after calcium deprivation as observed in intact rats. The 1,25-(OH)2D3 treatment increased the enzyme activity in uremic animals and resulted in an identical response to calcium deprivation as observed in intact rats; parathyroidectomy abolished this effect. A striking correlation between everted duodenal gut sac calcium transport and calcium ATPase activity could be demonstrated for all groups of rats studied. It is concluded that the calcium ATPase activity is linked to the production of 1,25-(OH)2D3 as well as to an additional factor, probably parathyroid hormone. The close relationship between enzyme activity and in vitro calcium transport, even during constant physiological supplementation with 1,25-(OH)2D3, suggests an autonomous role of the calcium ATPase activity for mediation of calcium transport in the duodenum in addition to the well-known mechanisms related to vitamin D and its metabolites.

scholarly journals In vitro fibrillogenesis of collagen II from pig vitreous humour

1995 ◽  
Vol 306 (3) ◽  
pp. 871-875 ◽  
Author(s):  
C Yang ◽  
H Notbohm ◽  
Y Açil ◽  
R Heifeng ◽  
S Bierbaum ◽  
...  
Keyword(s):  
Cross Sections ◽  
Soluble Fraction ◽  
Unit Length ◽  
Insoluble Fraction ◽  
Vitreous Humour ◽  
Collagen Types ◽  
Collagen Molecules ◽  
Collagen Ii

Collagen from pig vitreous humour was fractionated into a soluble and an insoluble fraction by centrifugation. Most of the collagen II in the soluble fraction was present as pN-collagen II (procollagen II without the C-terminal propeptide), besides smaller quantities of procollagen II, collagen II and two as yet unidentified alpha-chains of collagen II. Other collagen types may be present only in trace amounts. Collagen II of the insoluble fraction, which is mostly deposited in fibrillar aggregates, consists of both pN-collagen II and collagen II. To determine the possible role of collagen II precursors in the formation of the extracellular matrix of the vitreous humour these collagen molecules were purified and in vitro fibrillogenesis was used to demonstrate that pN-collagen II could form fibrils in mixtures with collagen II. These fibrils have a reduced mass per unit length depending on the content of pN-collagen in the mixture. Cross-sections of the newly formed fibrillar aggregates revealed a flattened shape. The incomplete processing of the precursors of collagen II may be part of regulatory mechanisms possibly controlling the formation of a translucent scaffold as is required in the vitreous humour.

scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

Characterization, Development and Localization of "Flavanone Synthase" in Tulip Anthers

1978 ◽  
Vol 33 (11-12) ◽  
pp. 841-846 ◽  
Author(s):  
R. Siitfeld ◽  
B. Kehrel ◽  
R. Wiermann
Keyword(s):  
Enzyme Activity ◽  
Gel Chromatography ◽  
Enzyme Release ◽  
Similar Data ◽  
Reaction Products ◽  
Isomerase Activity ◽  
Malonyl Coa ◽  
Specific Activities

1. “Flavanone synthase” was isolated from anthers of Tulipa cv. “Apeldoorn” and partially purified by (NH4) 2SO4 fractionation, gel chromatography and isoelectric focussing. The enzyme preparation was free of chalcone-flavanone isomerase activity. 2. p-Coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were found to be efficient substrates of the synthase. The products formed were naringenin (5,7,4′-trihydroxyflavanone), eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) and homoeriodictyol (5,7,4′-trihydroxy-3′-methoxyflavanone), respectively. Addition of thiol reagents at concentrations exceeding 10-3 м caused inhibition of the enzyme. “ Release products” , however, were not detectable. Although exclusively chalcones accumulate in the tulip anther, only flavanones but no chalcones were detectable in our in vitro system. 3. The apparent Km values for p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were 1 .7× 10-6 м, 1.6× 10-6 м and 2 .5 ×10-6 м, respectively. Similar data were observed for malonyl- CoA. 4. No cofactors are required for the synthase reaction. The enzyme is strongly inhibited by the reaction products flavanone and coenzyme A . Maximum enzyme activity was found at pH 8.0 and 30 °. The molecular weight was approx. 55,000. 5. Synthase activity develops in early postmeiotic stages of microsporogenesis. Highest specific activities of the enzyme coincide with a maximum in chalcone accumulation within the anthers. 6. The contents of anthers was separated into two fractions, pollen and tapetum. Highest specific activities were observed with tapetum fractions, while pollen fractions exhibited only very low activities. The high enzyme activity in the tapetum fraction points to the important role of the tapetum in the biosynthesis of flavonoids in the loculus of anthers.

scholarly journals Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

2001 ◽  
Vol 153 (5) ◽  
pp. 1061-1070 ◽  
Author(s):  
Shuh-ichi Nishikawa ◽  
Sheara W. Fewell ◽  
Yoshihito Kato ◽  
Jeffrey L. Brodsky ◽  
Toshiya Endo
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Elevated Temperatures ◽  
Carboxypeptidase Y ◽  
Mutant Form ◽  
Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

scholarly journals BmHSP20.8 is Localized in the Mitochondria and has a Molecular Chaperone Function In Vitro

2015 ◽  
Vol 15 (1) ◽  
pp. 99 ◽  
Author(s):  
Chengcheng Wu ◽  
Chan Wang ◽  
Dan Li ◽  
Yue Liu ◽  
Qing Sheng ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Chaperone Function

scholarly journals The role of metal oxide nanoparticles, Escherichia coli, and Lactobacillus rhamnosus on small intestinal enzyme activity

2020 ◽  
Vol 7 (12) ◽  
pp. 3940-3964
Author(s):  
Alba García-Rodríguez ◽  
Fabiola Moreno-Olivas ◽  
Ricard Marcos ◽  
Elad Tako ◽  
Cláudia N. H. Marques ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Metal Oxide Nanoparticles ◽  
Lactobacillus Rhamnosus ◽  
Engineered Nanomaterials ◽  
Oxide Nanoparticles ◽  
Small Intestinal ◽  
Intestinal Enzyme

To understand the effects of engineered nanomaterials added intentionally and unintentionally to food, we improved a gastrointestinal in vitro model using in vitro digested nanoparticles, Caco-2/HT29-MTX cells and gut microbiota.

Inhibition of intestinal degradation of somatostatin by rat milk

1990 ◽  
Vol 258 (3) ◽  
pp. G426-G431
Author(s):  
R. K. Rao ◽  
O. Koldovsky ◽  
T. P. Davis
Keyword(s):  
Small Intestine ◽  
Gastrointestinal Tract ◽  
Soluble Fraction ◽  
In Vitro Degradation ◽  
Weanling Rats ◽  
Gastric Lumen ◽  
Milk Casein ◽  
Soluble Fractions ◽  
Somatostatin 14

In vitro degradation of 125I-labeled somatostatin-14 (Tyr11) [I-SS-14(Tyr11)] by luminal flushings of rat gastrointestinal segments was studied to characterize the fate of somatostatin in the gastrointestinal lumen. In addition, we evaluated the effect of rat milk as a potential inhibitor of luminal degradation of 125I-SS-14(Tyr11). Degradation of 125I-SS-14(Tyr11) was not detected in stomach flushings from either suckling or weanling rats. Luminal flushings from the small intestine degraded 125I-SS-14(Tyr11), with a gradient increase of activity from duodenum to midjejunum (degradation in suckling rat midjejunum and ileum was about five times lower than that in weanling rat). Degradation of 125I-SS-14(Tyr11) by luminal flushings of suckling rat midjejunum was dose dependently inhibited by rat milk casein and soluble fractions. Inhibitory activity of rat milk soluble fraction was heat labile and several times more potent than that of casein fraction. Casein fraction appeared to be stable at 100 degrees C for up to 30 min of exposure. These studies suggest that somatostatin is stable in the gastric lumen and that milk protects somatostatin from intestinal luminal proteolysis, indicating a possible physiological significance of milk-borne SS-14 for the suckling rat gastrointestinal tract.

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