Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes.

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.

Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes.

Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis.

The use of porphyrins as probes to examine the requirement for hemin in the maintenance of protein synthesis in reticulocyte lysates.

Hemin was compared with other porphyrins for the ability to sustain protein synthesis in reticulocyte lysates and to inhibit the phosphorylation of the alpha subunit of initiation factor 2 (eIF-2 alpha). Iron-containing porphyrins (hemin, mesohemin IX, deuterohemin IX) were found to maintain protein synthesis while iron-deficient porphyrins (protoporphyrin IX, mesoporphyrin IX) were ineffective. All of the porphyrins and metalloporphyrins tested were observed to inhibit phosphorylation of eIF-2 alpha by highly purified hemin-controlled repressor, although at different concentrations. Phosphorylation of eIF-2 alpha was examined in reticulocyte lysates. In the absence of porphyrin, phosphorylation of eIF-2 was rapid and linear for the first 7 min, during which time protein synthesis was inhibited. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 alpha in the lysate during this period; however, hemin sustained protein synthesis whereas protoporphyrin IX did not. Thus, the requirement for iron-containing porphyrins in the maintenance of protein synthesis is not due strictly to an inhibition of the protein kinase activity of the hemin-controlled repressor but involves an alternative, although yet undeciphered, mode of action.

Cellular immune response in vitro: I. A requirement for time-dependent T-lymphocyte cell division of cytotoxic cells in the allogeneic response.

Immunocompetent lymphoid cells cultured in vitro with allogeneic stimulator cells have been shown to produce T-lymphocyte populations which are specifically cytotoxic in vitro to the stimulatory cells whether normal or malignant. Although the culture requirements as well as the allogeneic requirements are known, the events leading to the production of T-lymphocyte cytotoxic cells is poorly understood. This study examines the role of cell division in the production of allogeneic cytotoxic T-cells in vitro. The elimination of cell division during the first 24 hr of allogeneic culture does not affect the cell-mediated cytotoxic immune response in vitro. Cell division is required, however, from 24 hr through 96 hr in culture and not necessary after 96 hr.