ABSTRACT
Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt (+) agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·10(6). The corresponding data for the mt (-) agglutinin were 38 nm, 9.7 S and 1.3·10(6), respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.
ABSTRACT
Gametes of the unicellular green alga Chlamydomonas eugametos agglutinate via their flagella. The mating type plus agglutination factor was solubilized by relatively mild treatments such as a short pH shock or an osmotic shock indicating that it is an extrinsic membrane component. It was also extracted in the nonionic detergent Triton X-100. A simple two-step procedure consisting of gel filtration over Sepharose 4B-cross-linked followed by anion exchange chromatography of the void volume yielded an electrophoretically pure preparation of a single high molecular weight glycoprotein. The agglutination factor sedimented as a 9.3 S particle (assuming a density of 1.50) in sucrose gradients. This low value, compared with the high apparent molecular weight seen during gel filtration and electrophoresis, suggests that the agglutination factor is a rod-like molecule. This was confirmed by viewing rotary-shadowed preparations in the electron microscope. A population of long slender molecules was revealed (328 +/- 20 nanometers), many of which had a knob at one end and a flexible region about one fourth of the length from the other end.
ABSTRACT
The subcellular distribution of arabinogalactan protein (AGP) in etiolated bean hypocotyls was studied by isopycnic density centrifugation on sucrose gradients at different Mg(2+) concentrations. The distribution of hydroxyproline (a major amino acid in AGP) in the membrane-containing fractions indicated that hydroxyproline-containing proteins were associated with rough endoplasmic reticulum, possibly with the Golgi apparatus, and with the plasma membrane. Non-specific binding of hydroxyproline-containing molecules to membranes could be excluded. To detect AGPs, fractions obtained after isopycnic density centrifugation were isoelectrofocused on polyacrylamide gels, and the gels were stained with ß-Gal-Yariv reagent. Bands appeared only at low pH values, where also most hydroxyproline was found. In the fractions at low densities (presumably membranefree), several bands were visible supporting the idea of the heterogeneous character of soluble AGP. The distribution of AGP in the membranous fractions strongly indicated that AGP was associated with the plasma membrane. Specific agglutination of protoplasts in the presence of ß-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.
ABSTRACT
1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.
