Due to the dense organization of organelles, cytoskeletal elements, and protein complexes that make up the intracellular environment, it is likely that membrane-permeant solutes may be excluded from a fraction of the interstitial space of the cytoplasm via steric restrictions, electrostatic interactions, and other long-range intermolecular forces. This study investigates the hypothesis that the intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery in response to hyperosmotic loading, based on prior theoretical and biomimetic experimental studies. Osmotic loading experiments are performed on immature bovine chondrocytes using culture conditions where regulatory volume responses are shown to be insignificant. Osmotic loading with membrane-permeant glycerol (92 Da) and urea (60 Da) are observed to produce partial volume recoveries consistent with the proposed hypothesis, whereas loading with 1,2-propanediol (76 Da) produces complete volume recovery. Combining these experimental results with the previous theoretical framework produces a measure for the intracellular partition coefficient of each of these solutes. At 1000 mOsm, 1,2-propanediol is the only osmolyte to yield a partition coefficient not statistically different from unity, kappa(p)(i) = 1.00 +/- 0.02. For glycerol, the partition coefficient increases with osmolarity from kappa(p)(i) = 0.48 +/- 0.19 at 200 mOsm to kappa(p)(i) = 0.80 +/- 0.07 at 1000 mOsm; urea exhibits no such dependence, with an average value of kappa(p)(i) = 0.87 +/- 0.07 for all osmolarities from 200 to 1000 mOsm. The finding that intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery under osmotic loading offers a simple method for characterizing the partition coefficient. These measurements suggest that significant partitioning may occur even for small membrane-permeant osmolytes. Furthermore, a positive correlation is observed, suggesting that a solute's cytoplasmic partition coefficient increases with increasing hydrophobicity.

We isolated gamma-glutamyltransferase [(gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2] from human liver and compared some of its properties with the same enzyme prepared from human kidney. The enzymes from these two sources are very similar with respect to initial velocity kinetic constants, pH optima of the transpeptidation and autotransfer reactions, heat stability, competitive inhibition by glutathione of the colorimetric assay in which gamma-glutamyl-4-nitroanilide is substrate, stability of catalytic activity to trypsinization, and relative rates of transfer of the gamma-glutamyl moiety from gamma-glutamyl-4-nitroanilide and L-[glycine-2-3/]glutathione to some amino acids and small peptides. The kidney enzyme is inhibited more by the gamma-glutamyl acceptor substrate, glycylglycine, as reflected in a sevenfold lower value for the inhibition constant KiA. Major differences were observed in the lectin-binding properties of liver gamma-glutamyltransferase compared to the kidney enzyme. Lectin-binding property differences are retained for the trypsinized form of the liver and kidney enzymes, although the degree of precipitation was less for certain lectins as compared to the untreated enzyme. Lectin-binding properties were reversed by carbohydrates specific for each lectin. We adapted the histochemical staining technique of Rutenberg et al. [J. Histochem. Cytochem. 17, 517 (1969)] to the detection of gamma-glutamyltransferase activity in acrylamide gels; diffusion artifacts are minimized and the color produced is stable for several days. Untreated and trypsinized forms of the liver enzyme both migrated faster in acrylamide gels (as single bands) than did the corresponding forms of the kidney enzyme.