Binding of an opiate, levorphanol, to intact neuroblastoma cells in continuous culture.

Mouse neuroblastoma cells in continuous culture, incubated for 1 to 2 days in the presence of 10--6 M levorphanol or morphine, were found to become tolerant to and dependent on those biologically active opiates. Examination of the interaction between levorphanol and the whole neuroblastoma cell suggested that levorphanol was binding to stereospecific opiate receptor sites. This binding was time and temperature dependent, and saturable at concentrations greater than 10--5 M levorphanol. Competition by other opiates for levorphanol sites correlated with their biological activity. This is the first evidence for saturable, stereospecific opiate binding in a homogeneous population of unhybridized cells in continuous culture.

Evidence for nuclear sites of stereospecific opiate binding in neuroblastoma cells in continuous culture.

A class of stereospecific, saturable receptors for narcotic drugs has been found in a clone of mouse neuroblastoma cells in continuous culture. Cells grown in the presence of 10(-8) M etorphine, a synthetic opiate, had an increased doubling time. The neuroblastoma cell nucleus was found to be the sole site for the high affinity binding of etorphine. It was found that these nuclear receptors (1) became saturated at a concentration of 2 X 10(-9) M etorphine, (2) had a dissociation constant of 5 X 10(-11) M etorphine, (3) were capable of binding etorphine stereospecifically at 37 degrees C but not at 4 degree C, and t4) were protein in nature as evidenced by treatment with proteolytic enzymes. More importantly, the receptor activity appeared to be chromatin-associated.

Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells.

Nascent DNA-nuclear membrane complexes isolated from HeLa cells and solubilized in a sodium dodecyl sulfate-urea solution were examined by gel electrophoresis, column chromatography, isopycnic centrifugation, and by extraction with chloroform/methanol. Radioactivity attributable to [3H]DNA co-migrated with three protein peaks during electrophoresis. This radioactivity was eliminated by prior treatment with DNAase. In addition, all of the radioactivity attributable to nascent DNA eluted with a specific protein on Sepharose 4B columns. This DNA - protein complex banded at a density of 1.58 gm/cm3 in sucrose-CsCl gradients. Treatment with DNAase, phospholipase A and C, and dilute alkali disrupted the complex. Moreover, 93% of the radioactivity attributable to protein and 70% of that attributable to DNA could be extracted from the complex with a chloroform/methanol solution. The results suggest that nascent DNA may be in a stable association with a proteolipid moiety of the nuclear membrane.

S-phase dependent forms of DNA - nuclear membrane complexes in HeLa cells.

DNA - nuclear membrane complexes were isolated from HeLa cells and examined by either zone sedimentation analysis or isopycnic centrifugation in sucrose/CsCl gradients. The data suggest that the complexes formed during the first 10 min of the S-phase remain as stable structures throughout the cell cycle. Other DNA - nuclear membrane complexes are formed at later times during replication. These later complexes appear as multiple species and the association of DNA and the nuclear membrane seems to be of a transient nature. Together, these results suggest that both the replicative origins and the replication points of the DNA are associated with the nuclear membrane. Although the complexes formed at the start of the S-phase and at later times during the S-phase appear to differ, these differences may provide them with the needed properties to serve as spatial organizers for the temporal regulation of DNA replication.

On the character of the nuclear membrane-associated DNA of HeLa cells.

The nature of the nascent DNA-membrane complexes isolated from synchronized HeLa cells was examined. It was found that membrane-associated DNA is not the result of repair replication. A study of the stability of the complexes to various degradative agents and enzymes suggest that the DNA is associated with a structure which is composed of a lipoprotein. An examination of the physical structure of this DNA indicates that it might be a "nicked" duplex with a base content similar to that of the bulk DNA.