Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel from L-Ala. [3H]Gly and D-Ala.[3H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg2+. The microvilli vesicles transiently accumulated radiolabel from L-Ala. [3H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular greater than intravesicular) Na+ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all 3H uptake from L-Ala. [3H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate of L-Ala.[3H]Gly hydrolysis to free alanine and [3H[glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel from L-Ala.[3H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate that L-Ala.[3H]Gly is hydrolyzed at the external surface of the microvilli with the [3H]glycine released being transported into the vesicles by a Na+ gradient-dependent system identical to that employed for free glycine. Microvilli vesicle uptake of radiolabel from D-Ala.[3H]Gly exhibited no Na+ dependent "over-shoot" effect. D-Ala.[3H]Gly was completely resistant to microvilli-catalyzed hydrolysis. Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range of L-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli. Renal dipeptidase catalyzed hydrolysis of L-Ala.Gly but not D-Ala.Gly, as was the case with microvilli-catalyzed hydrolysis of the dipeptides. With its location in the renal brush border microvilli and its hydrolytic action against L-dipeptides, renal dipeptidase my act at the luminal surface of the proximal tubule cell to hydrolyze L-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na+ gradient-dependent processes.
Minimal deviation hepatoma 7288C cells (HTC) were incubated in serum-supplemented and serum-free Swim's 77 medium in the presence of D-[1-14C] glucose for 1, 2, 4, 8, 12 and 24 hr. Glucose oxidation to CO2, incorporation into total cell mass, and incorporation into cell and medium lipids were determined. The percentage distribution of total cell lipid radioactivity in individual neutral and polar lipid classes was followed as a function of time. Degradation studies of individual lipid classes were performed to ascertain the percentage of radioactivity in acyl and glycerol moieties. The percentage of D-[1-14C] glucose oxidized to 14CO2, incorporated into cell matter and cell lipids was elevated in cells incubated in serum-free medium as opposed to serum-supplemented medium. The percentage distribution of total cell lipid radioactivity into individual neutral lipid classes from both serum-free and serum-supplemented cultures was as follows: sterols greater than triglycerides greater than free fatty acids greater than sterol esters. The percentage distribution of total cell lipid radioactivity into individual polar lipid classes of serum-supplemented cultures was as follows: phosphatidylcholine greater than phosphatidylinositol greater than sphingomyelin greater than phosphatidylethanolamine greater than phosphatidylserine. The distribution of glucose radiolabel into individual polar lipid classes of serum-free HTC cells was different from their serum-supplemented counterparts: sphingomyelin greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylserine. Glycerol from glyceride classes contained a higher percentage of radioactivity than the acyl moieties, with this percentage significantly elevated in serum-free cultures. The data indicate that, although glucose is a substrate for HTC cell lipids, other precursors present in the culture system also contribute to the lipid constituency of this hepatoma cell line.
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