scholarly journals Increased hyaluronate synthesis is required for fibroblast detachment and mitosis

1986 ◽  
Vol 239 (2) ◽  
pp. 445-450 ◽  
Author(s):  
M Brecht ◽  
U Mayer ◽  
E Schlosser ◽  
P Prehm
Keyword(s):  
Ion Exchange ◽  
Human Embryo ◽  
Culture Medium ◽  
Glucuronic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cellular Fraction ◽  
Embryo Fibroblasts ◽  
Hyaluronate Synthase

Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.

scholarly journals Isolation of streptococcal hyaluronate synthase

1986 ◽  
Vol 235 (3) ◽  
pp. 887-889 ◽  
Author(s):  
P Prehm ◽  
A Mausolf
Keyword(s):  
Ion Exchange ◽  
Glucuronic Acid ◽  
Cetylpyridinium Chloride ◽  
Active Form ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Triton X ◽  
Hyaluronate Synthase

Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.

scholarly journals Structure of heparin-derived tetrasaccharides

1985 ◽  
Vol 229 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Z M Merchant ◽  
Y S Kim ◽  
K G Rice ◽  
R J Linhardt
Keyword(s):  
Biological Activity ◽  
Ion Exchange ◽  
Fast Atom Bombardment ◽  
Glucuronic Acid ◽  
Anticoagulant Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Spectroscopic Methods ◽  
Iduronic Acid

The structure of heparin was examined by characterizing a disaccharide and five of the more than a dozen tetrasaccharide components obtained by its depolymerization with flavobacterial heparinase. Enzymic depolymerization of porcine mucosal heparin results in a mixture of di-, tetra-, hexa- and higher oligo-saccharides. The di- and tetra-saccharide components represent 75mol/100mol of these heparin fragments. Ion-exchange chromatography indicates the presence of only one disaccharide, deltaIdu2S(1→4)-alpha-D-GlcNS6S (where Idu is iduronic acid, deltaIdu is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, GlcA is glucuronic acid and S is sulphate), but results in the isolation of five major and at least seven minor tetrasaccharide components. The structures of the disaccharide and five major tetrasaccharides were determined by chemical, enzymic, electrophoretic and spectroscopic methods, including 13C, 1H n.m.r. and fast atom bombardment-m.s. The structure of these five tetrasaccharides are: delta Idu2S(1→4)-alpha-D-GlcNS6S(1→4)-alpha-L-Idu2S(1→4)-alpha -D-GlcNS6S; delta Idu2S(1→4)-alpha-D-GlcNS6S(1→4)-beta-D-GlcA(1→4)- alpha-D-GlcNS6S; delta Idu2S(1→4)-alpha-D-GlcNS(1→4)-beta-D-GlcA delta Idu2S(1→4)-alpha-D-GlcNAc(1→4)-beta-D-GlcA(1→4)- alpha-D-GlcNS6S; and delta Idu2S(1→4)-alpha- D-GlcNAc(1→4)-alpha-L-Idu(1→4)-alpha-D-GlcNS6S. Biological activity for the disaccharide and the five major tetrasaccharides was examined, and none of them were found to possess significant anticoagulant activity.

scholarly journals Biosynthesis of dermatan sulphate. Loss of C-5 hydrogen during conversion of d-glucuronate to l-iduronate

1981 ◽  
Vol 198 (3) ◽  
pp. 669-675 ◽  
Author(s):  
A Malmström
Keyword(s):  
Ion Exchange ◽  
Paper Chromatography ◽  
Glucuronic Acid ◽  
Human Fibroblasts ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dermatan Sulphate ◽  
Microsomal Preparation ◽  
Iduronic Acid

The formation of L-iduronic acid during biosynthesis of dermatan sulphate has been studied in culture human fibroblasts and in microsomes from the same cells. The cells were incubated with D-[14C]glucose and D-[5-3H]glucose for 72 h. The [14C,3H]dermatan sulphate was hydrolysed and the disaccharides obtained were acetylated and separated by ion-exchange chromatography. The ratio of 3H/14C was 0.36 for N-acetyldermosine and 1.36 N-acetylchondrosine. A microsomal preparation from the fibroblasts was incubated with UDP-D-[5-3H]glucuronic acid, UDP-D-[14C]glucuronic acid, UDP-N-acetyl-D-galactosamine and 3′-phospho-5′-adenylyl sulphate. The polymeric products were separated into nonsulphated and sulphated components which had 3H/14C ratios of 0.51 and 0.20 and contained 9% and 70% of their uronosyl residues in the L-ido-configuration, respectively. Chondroitinase-AC digestion of these polymers liberated all of the remaining 3H activity. Hydrolysis and N-acetylation followed by paper chromatography showed that the L-iduronic acid-containing products were devoid of 3H. The data obtained indicate that the epimerization of D-glucuronosyl to L-iduronosyl residues during biosynthesis of dermatan sulphate involves an abstraction of the C-5 hydrogen of the uronosyl residue.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

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