Intracellular hyaluronan in arterial smooth muscle cells: association with microtubules, RHAMM, and the mitotic spindle.

Although considered a pericellular matrix component, hyaluronan was recently localized in the cytoplasm and nucleus of proliferating cells, supporting earlier reports that hyaluronan was present in locations such as the nucleus, rough endoplasmic reticulum, and caveolae. This suggests that it can play roles both inside and outside the cell. Hyaluronan metabolism is coupled to mitosis and cell motility, but it is not clear if intracellular hyaluronan associates with cytoskeletal elements or plays a structural role. Here we report the distribution of intracellular hyaluronan, microtubules, and RHAMM in arterial smooth muscle cells in vitro. The general distribution of intracellular hyaluronan more closely resembled microtubule staining rather than actin filaments. Hyaluronan was abundant in the perinuclear microtubule-rich areas and was present in lysosomes, other vesicular structures, and the nucleolus. Partially fragmented fluorescein-hyaluronan was preferentially translocated to the perinuclear area compared with high-molecular-weight hyaluronan. In the mitotic spindle, hyaluronan colocalized with tubulin and with the hyaladherin RHAMM, a cell surface receptor and microtubule-associated protein that interacts with dynein and maintains spindle pole stability. Internalized fluorescein-hyaluronan was also seen at the spindle. Following telophase, an abundance of hyaluronan near the midbody microtubules at the cleavage furrow was also noted. In permeabilized cells, fluorescein-hyaluronan bound to RHAMM-associated microtubules. These findings suggest novel functions for hyaluronan in cellular physiology.     

Two-color immunofluorescence using a fluorescence-activated cell sorter.

A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.     

Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases.

In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.     

Sterol 24(28) methylene reductase in Saccharomyces cerevisiae.

Optimal conditions for the 24(28)methylene reductase were obtained. The enzyme assay provided for unusually high activity; the Km was determined to be 10.8 mum. The enzyme activity was increased in cells grown with ethanol as the substrate.