Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

2018 ◽  
Vol 35 (2) ◽  
pp. e2768 ◽  
Author(s):  
Sabrina Ait Braham ◽  
Fouzia Hussain ◽  
Roberto Morellon‐Sterling ◽  
Shagufta Kamal ◽  
Jakub F. Kornecki ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Proteolytic Activity ◽  
Covalent Attachment ◽  
Enzyme Stabilization

scholarly journals Studies of the active site of m-calpain and the interaction with calpastatin

1993 ◽  
Vol 296 (1) ◽  
pp. 135-142 ◽  
Author(s):  
C Crawford ◽  
N R Brown ◽  
A C Willis
Keyword(s):  
Ion Exchange ◽  
Proteolytic Activity ◽  
Active Site ◽  
Binding Assay ◽  
Large Subunit ◽  
Gel Filtration ◽  
Small Subunit ◽  
Binding Domain ◽  
Competitive Binding Assay ◽  
Binding Domains

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

Embedding Procedure for Water Swollen Samples

1970 ◽  
Vol 28 ◽  
pp. 504-505
Author(s):  
Ann M. Thomas ◽  
Virginia Shemeley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ion Exchange ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Exchange Resins ◽  
Transmission Electron ◽  
First Pass ◽  
Exchange Resins

Those samples which swell rapidly when exposed to water are, at best, difficult to section for transmission electron microscopy. Some materials literally burst out of the embedding block with the first pass by the knife, and even the most rapid cutting cycle produces sections of limited value. Many ion exchange resins swell in water; some undergo irreversible structural changes when dried. We developed our embedding procedure to handle this type of sample, but it should be applicable to many materials that present similar sectioning difficulties.The purpose of our embedding procedure is to build up a cross-linking network throughout the sample, while it is in a water swollen state. Our procedure was suggested to us by the work of Rosenberg, where he mentioned the formation of a tridimensional structure by the polymerization of the GMA biproduct, triglycol dimethacrylate.

Sem Observations in Gastric Mucosa Exposed to Progressive Enzymatic Etching

1980 ◽  
Vol 38 ◽  
pp. 570-571
Author(s):  
C.A.E. Lemmi ◽  
D. Booth ◽  
G.E. Adomian
Keyword(s):  
Gastric Mucosa ◽  
Critical Point ◽  
Proteolytic Activity ◽  
Etching Process ◽  
Cellular Types

In order to enrich populations of homogeneous cellular types we dissociated gastric mucosa by enzymatic techniques. In addition, we used SEM to monitor the progressive etching of the mucosa. Two enzymes were tested: collagenase III with minimum proteolytic activity and Pronase with broader proteolytic effects. The gastric mucosa was exposed to the effect of the enzymes using everted stomach preparations. In this way the digestive action occured progressively from the lumen of the stomach toward the base of the glands. This “etching” process could be monitored conveniently by SEM. After incubation for periods varying from 30 to 210 minutes the tissues were stretched on dental wax, fixed in 2 % glutaralheyde, post-fixed in osmium, dehydrated, critical point dryed and coated with gold. A model MSM-5 “Mini-SEM” was used for observation. Gentle uncurling of the preparation before coating with gold produced fractures which revealed the structure of the gastric glandsin more detail.

SYNTHESIS AND MAGNETIC PROPERTIES OF PLATELET SHAPE SPINEL AND M-TYPE HEXAGONAL FERRITES PREPARED FROM β'' ALUMINA-LIKE FERRITES BY ION EXCHANGE

1988 ◽  
Vol 49 (C8) ◽  
pp. C8-937-C8-938
Author(s):  
O. Kalogirou ◽  
A. C. Stergiou ◽  
D. Samaras ◽  
S. Nicolopoulos ◽  
A. Bekka ◽  
...  
Keyword(s):  
Magnetic Properties ◽  
Ion Exchange ◽  
Hexagonal Ferrites ◽  
Platelet Shape
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