Human Spleen Tissue Research Articles

Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP

Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.

The effect of a heat-stable factor in human spleen on glucocerebrosidase and acid β-glucosidase activities

As has been reported earlier [Ho, M. W. and O'Brien, J.S. (1971), Proc. Natl. Acad. Sci. U.S. 68, 2810–2813], a heat-stable factor is present in normal human spleen and in tissue samples from patients with Gaucher's disease which enhances acid β-glucosidase activity as measured with the fluorogenic substrate 4-methylumbelliferyl-β- d-glucopyranoside. Glucocerebrosidase activity is not increased by the addition of as much as 8 times the quantity of heat stable factor normally present in human spleen tissue. Glucocerebrosidase activity (5000 units/g of spleen tissue) is greatly in excess of the heat-stable β-glucosidase stimulating factor (200 units/g spleen). Consequently, it is unlikely that the factor plays a physiologically significant role in the metabolism of glucocerebroside.

Opportunity to Advance Knowledge of Gaucher's Disease

We are interested in the metabolic aberrations which occur in patients with sphingolipidoses and in particular in Gaucher's disease. We have recently synthesized some glucoselabeled cerebroside and have examined the catabolism of this compound in normal rat and human spleen tissues. We have found a very active enzyme which splits this material in these tissues and we are particularly interested in examining spleen tissues from Caucher's patients to try to determine if this particular enzyme is present or absent in tissues from these patients.