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1. A crude lysosomal fraction obtained by differential centrifugation of a rat liver homogenate was subjected to zonal centrifugation in iso-osmotic self-generating gradients composed of modified colloidal silica (Percoll). Analysis of relevant marker-enzyme activities shows a continuous band of considerably purified lysosomal particles in the density range 1.04--1.11 g/ml. 2. A relationship between age and buoyant density of the parenchymal lysosomal subpopulations is indicated by the distribution of 125I-labelled asialoglycoproteins in the heterogeneous lysosomes during the catabolism of the glycoprotein. The labelled asialoglycoprotein first appeared in lysosomal particles of low density, which with time progressively acquired a higher density. Furthermore, 30 min after administration the 125I-labelled asialocaeruloplasmin recovered in the light lysosomes was less degraded than the material recovered in the heavy lysosomes. 3. A lysosomal enzyme (arylsulphatase) was found to possess considerably higher isoelectric points in the heavy lysosomes than in the light lysosomes, which is consistent with a relationship between age and density of the lysosomes.

A free-diffusion method has been developed for the determination of the intradiffusion coefficient ('self-diffusion coefficient') of a polymer in highly concentrated solutions. A fraction of the polymer is labelled with a small amount of light-absorbing substituent. The diffusion of this labelled species, present in low concentration, is followed in the presence of a high concentration of unlabelled material with the aid of absorption optics in the analytical ultracentrifuge. The diffusion proceeds over a boundary at which the difference in concentration of unlabelled material is varied. The average concentration of total polymer and the concentration difference of the labelled material are, however, constant. From theoretical considerations it is shown that by extrapolation of the diffusion coefficient so obtained to zero concentration difference of total material, the intradiffusion coefficient of the polymer at that concentration is obtained. The procedure also permits the ordinary translational diffusion coefficient to be estimated. The method has been applied to two dextran fractions with weight-average molecular weights of 19000 and 150000, which were labelled with fluorescein groups. As expected, the intradiffusion coefficient decreases with increasing polymer concentration, the decrease being more pronounced for the high-molecular-weight material. This decrease in the diffusion rate of dextran is, however, less than the corresponding decrease in the sedimentation rate which proteins with similar hydrodynamic parameters experience in dextran solutions. This agrees with the hypothesis that flexible linear polymers move through a network as chains rather than as hydrodynamic spheres. By combining measurements of the ordinary diffusion coefficient and the intradiffusion coefficient, it is possible to calculate the thermodynamic properties (as expressed by the virial expansion) of the system. This method is of particular importance in studies on concentrated solutions of high-molecular-weight polymers.The intercellular matrix of connective tissues contains high-molecular-weight polysaccharides (glycosaminoglycans) anchored to proteins [I]. The concentration of these polysaccharides varies but may in some tissues, e.g. cartilage, be as high as 5 -10% (w/v). Marked concentration differences of polysaccharide exist between different compartments.The physiological functions of the glycosaminoglycans have been related to the physicochemical properties of the concentrated polysaccharide solutions [2,3]. It has for instance been postulated that the connective tissues form diffusion barriers because the presence of polysaccharides increases the frictional resistance to transport of other macromolecules [4]. This effect has been studied in detail in recent sedimentation and diffusion investigations [5 -81. This paper is no. 25 in the series Interaction between Polysaccharides and other Macromolecules.Abbreviufiuns. Dx-I 9 and Dx-150, dextrans with weightaverage molecular weights of approximately 19000 and 1 SO000 resp...

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To investigate the role of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A, point mutated variants of the protein were stably expressed in the beta1-deficient cell line GD25. Mutants T777A, Y783F, S785A, and Y795F were fully active in promoting cell adhesion, de novo formation of focal contacts, formation of fibronectin fibrils, and activation of focal adhesion kinase. Thus, phosphorylation of these residues is not required for several basic functions of integrin beta1A. On the other hand, the TT788-9AA mutant, was defective in mediating cell attachment and did not contribute to fibronectin fibril formation. The conformation of the extracellular domain was shifted towards an inactive state as measured by binding of the monoclonal antibody 9EG7. Antibody induced clustering of beta1ATT788-9AA demonstrated that the mutant cytoplasmic part was functional in mediating activation of focal adhesion kinase. Therefore, we conclude that threonines 788–789, which are conserved among most integrin beta subunits, are of critical importance for integrin function due to effects on the extracellular conformation of the receptor.