Isolation of a ß-galactoside-binding lectin from cat liver

Abstract

A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing ß-galactosyl residues, of which the 1-amine-1-deoxy-ß-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM ß-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble ß-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.

Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots

“…The HRP interaction with Con A-Sepharose has demonstrated to be highly dependent of the ligand density [9]. The results herein reported indicate that in order to adsorb more than 90% of added HRP, Con A-derivatives must contain not less than 22 mg of immobilized lectin/ml of packed gel.…”

“…Aliquots (3.0 g) of suction-dried CDAP-activated Sepharose equilibrated in coupling buffer were incubated with 3.0 ml of solutions containing increasing amounts of pure Con A dissolved in coupling buffer, and mixed end over end at room temperature for 4 h as described in [9]. The Con A-gel derivatives were washed on a glass filter with coupling buffer, distilled water and finally with 0.1 M acetate buffer pH 6.0 containing 0.5 M NaCl, 1 mM CaCl 2 and 1 mM MnCl 2 (adsorption buffer).…”

A New Galactose-Specific Lectin from Clerodendrum infortunatum

“…For further purification of the protein, the pooled protein fractions showing haemagglutination, obtained after gel filtration were subjected to the affinity chromatography with galactose coupled Sepharose-4B as per the protocol of Franco-Fraguas et al (27) with slight modifications. Sepharose-4B (20 g) was incubated in 0.4 M NaOH and 5% epichlorohydrin at 40 °C for 2 hours.…”

Purification and some properties of galectin‐1 derived from water buffalo (Bubalus bubalis) brain

“…The predominant forms of galectins are dimers, whereas sponge galectins have been proposed to form trimers and or tetramers. The most common and abundant galectin in mammalian tissues such as muscle, heart, lung, placenta, spleen, brain and small intestine is galectin-1 which has subunit molecular weight very close to 14 kDa (Clerch et al, 1988;Ahmed et al, 1996;Ola et al, 2001;Franco-Fraguas et al, 2003). The majority of galectins have a highly conserved carbohydrate binding site with affinity for lactose and N-acetyllactosamine (Leffler and Barondes, 1986;Ahmed and Vasta, 1994).…”