Interfacial catalysis by phosphoinositide 3'-hydroxykinase.

Phosphorylation of phosphoinositides by phosphoinositide 3'-hydroxykinase (PI3K) occurs at a lipid/water interface. We have determined that highly purified recombinant human P13K binds tightly to vesicle interfaces composed primarily of phosphatidylinositol (PI) or 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM). The rate of desorption of PI3K from the vesicle interface is slow and does not significantly affect the observed product formation kinetics. Observations which demonstrate that PI3K is tightly bound to the vesicle lipid/water interface include the following: (1) product formation plateaus rapidly, even in the presence of active enzyme and excess substrate; (2) total product formation is proportional to the amount of PI3K; (3) initial product formation rates are unaffected by bulk lipid concentration but are dependent on the interfacial substrate concentration; and (4) PI3K partitions with lipid vesicles in sedimentation gradients. This enzymatic profile has been referred to as catalysis in the "scooting" mode (Berg et al., 1991). A kinetic analysis of PI3K catalysis in the scooting mode is presented. The interfacial Km,app for PI was determined to be approximately 6.0 mol % in PI/DMPM vesicles. The ratio of specificity constants (kcat/Km) for PI, phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-diphosphate (PIP2) utilization was determined to be near unity. These results provide a rigorous enzymological framework for the kinetic analysis of PI3K inhibitors.

Separation of ovine chromaffin granules from lysosomes by successive isoosmolar and hyperosmolar density gradient centrifugation.

Preparations containing ovine chromaffin granules and lysosomes were obtained by differential centrifugation and applied to density gradients. In isoosmolar linear Metrizamide gradients the granules had a lower density than the major portion of the lysosomes (1.05 compared to 1.15); however, in hyperosmolar linear Metrizamide gradients the granule density increased and they migrated close to the lysosomes. The granules separated into two bands on a discontinuous isoosmolar Metrizamide gradient; however, these two bands were similar in terms of granule and lysosomal markers. On a discontinuous hyperosmolar sucrose gradient the granules were more dense than the lysosomes, the reverse of the situation on the Metrizamide gradient. Separation on a discontinuous isoosmolar Metrizamide gradient followed by a 1.8 M sucrose cushion provided a 54-fold purification of granules from lysosomes and similar separations from other subcellular markers. This procedure also provided a 37-fold purification of bovine granules from lysosomes. It was demonstrated that thimet oligopeptidase (EC 3.4.24.15) occurred in the adrenal medulla but is not principally located in the chromaffin granule.

Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles.

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

Isopycnic centrifugation of sheared L-cell chromatin in metrizamide gradients.

Isopycnic gradient centrifugation of L-929 cell chromatin in a 38% (W/V) metrizamide solution yields two distinct fractions. The fraction banding at a density of 1.24 gm/cm(2) (H chromatin) contains about 10% of the DNA present in the fraction banding at a density of 1.18 gm/cm(2) (L chromatin). Both fractions contain the same proportions of satellite to main band DNA's. Some differences can be seen in the DNA: protein ratios and types of proteins present in the H and L chromatin fractions.