scholarly journals The synthesis and purification of 2′-carboxy-d-arabinitol 1-phosphate, a natural inhibitor of ribulose 1,5-bisphosphate carboxylase, investigated by31P n.m.r

1989 ◽  
Vol 260 (3) ◽  
pp. 711-716 ◽  
Author(s):  
S Gutteridge ◽  
G S Reddy ◽  
G Lorimer
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzymic Hydrolysis ◽  
Bisphosphate Carboxylase ◽  
Natural Inhibitor

2′-Carboxy-D-arabinitol 1-phosphate (2CA1P), a natural inhibitor of ribulose 1,5-bisphosphate carboxylase was synthesized from 2′-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The selective dephosphorylation of 2CABP with either acid phosphatase or alkaline phosphatase was investigated by using 31P n.m.r. The n.m.r. spectra of the progress of the reactions indicated that both phosphatases preferentially removed the 5-phosphate from the bisphosphate. After the consumption of all of the bisphosphate, alkaline phosphatase generated a mixture of 2′-carboxy-D-arabinitol 1- and 5-monophosphates in the ratio of about 4:1, along with Pi. The enzyme also hydrolysed the monophosphates to 2′-carboxyarabinitol, thus decreasing the yield of 2CA1P further. In contrast, acid phosphatase catalysed almost quantitative conversion of 2CABP into 2CA1P, preferring to hydrolyse only the 5-phosphate. In either case, separation of the 2CA1P from Pi or other products of enzymic hydrolysis was readily accomplished by conventional ion-exchange chromatography or h.p.l.c.

LINGCOD MUSCLE PHOSPHORIBOISOMERASE AND RIBULOSE 5′-PHOSPHATE 3′-EPIMERASE

1959 ◽  
Vol 37 (8) ◽  
pp. 961-973 ◽  
Author(s):  
H. L. A. Tarr
Keyword(s):  
Acid Phosphatase ◽  
Ion Exchange ◽  
Ammonium Sulphate ◽  
Simple Procedure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Extraction ◽  
Enzyme Preparations

Comparatively pure phosphoriboisomerase and ribulose 5′-phosphate 3′-epimerase enzyme preparations were obtained from lingcod muscle by a simple procedure involving water extraction, saturation of the extract with ammonium sulphate, dialysis, brief heating to 55 °C, lyophilization of the solution, and final separation by ion exchange chromatography, using diethylamiuoethyl cellulose columns. Both enzymes have broad pH optima above pH 7.0, but are rapidly inactivated below this value. The following equilibria were established and compared with those obtained by other investigators: [Formula: see text], 1.35:1.0; [Formula: see text], 1: 1.5 and [Formula: see text], 1:0.58:0.66. The ketopentulose phosphate resulting from the action of phosphoriboisomerase on D-ribose 5-phosphate was isolated and identified as D-ribulose5-phosphate. Both D-ribulose and D-xylulose were demonstrated after subjecting a product of epimerase action to hydrolysis by acid phosphatase and ion exchange chromatography.

LINGCOD MUSCLE PHOSPHORIBOISOMERASE AND RIBULOSE 5′-PHOSPHATE 3′-EPIMERASE

1959 ◽  
Vol 37 (1) ◽  
pp. 961-973
Author(s):  
H. L. A. Tarr
Keyword(s):  
Acid Phosphatase ◽  
Ion Exchange ◽  
Ammonium Sulphate ◽  
Simple Procedure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Extraction ◽  
Enzyme Preparations

Comparatively pure phosphoriboisomerase and ribulose 5′-phosphate 3′-epimerase enzyme preparations were obtained from lingcod muscle by a simple procedure involving water extraction, saturation of the extract with ammonium sulphate, dialysis, brief heating to 55 °C, lyophilization of the solution, and final separation by ion exchange chromatography, using diethylamiuoethyl cellulose columns. Both enzymes have broad pH optima above pH 7.0, but are rapidly inactivated below this value. The following equilibria were established and compared with those obtained by other investigators: [Formula: see text], 1.35:1.0; [Formula: see text], 1: 1.5 and [Formula: see text], 1:0.58:0.66. The ketopentulose phosphate resulting from the action of phosphoriboisomerase on D-ribose 5-phosphate was isolated and identified as D-ribulose5-phosphate. Both D-ribulose and D-xylulose were demonstrated after subjecting a product of epimerase action to hydrolysis by acid phosphatase and ion exchange chromatography.

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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