Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A. Requirement for threonines 788-789 in receptor activation.

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.

cDNA cloning and sequencing of rat alpha 1-macroglobulin.

cDNA clones coding for the plasma protease inhibitor alpha 1-macroglobulin were isolated from a rat liver library. The obtained cDNA sequence contained 4701 nucleotides and had an open reading frame coding for a 1,500 amino acid long protein, including a 24 amino acid signal peptide. The identity of the deduced protein sequence as alpha 1-macroglobulin was established by comparison with published peptide sequences of the protein. The mature protein shares 53% and 57% overall amino acid identity with the two other identified members of the rat alpha-macroglobulin family, alpha 1-inhibitor 3 and alpha 2-macroglobulin. A sequence typical for an internal thiol ester was identified. Of the 24 cysteines, 23 are conserved with alpha 2-macroglobulin. However, instead of the two most C-terminal cysteines in alpha 2-macroglobulin, which forms a disulfide bridge in the receptor binding domain, alpha 1-macroglobulin contains phenylalanine. One mRNA species hybridizing with the alpha 1-macroglobulin probe was observed in rat and mouse liver RNA (approximately 6.2 kb), whereas no corresponding transcript was detected in RNA from human liver.

Separation of human monocytes on density gradients of Percoll.

Monocytes from human blood have been isolated by centrifugation in Percoll. A one-step procedure has been designed to isolate the cells from 7 ml of blood. when 5% of the white blood cells are assumed to be monocytes, an estimated average yield of 100% and a purity of 20% is achieved. The contaminating cells are almost exclusively lymphocytes. By a two-step procedure the monocytes can be obtained 90% pure with an approximate yield of 35%. The cells can be used for tissue culture without washing and they display the usual properties of mononuclear phagocytes in vitro.

Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation.

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.

Diffusion of dextran in concentrated solutions.

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 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.