Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts.

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 X 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.     

Subcellular compartmentalization of cAMP-dependent protein kinase regulatory subunits during palate ontogeny

Mammalian palatal ontogeny involves epithelial-mesenchymal interactions, cell differentiation, and cell movements. These events occur on days 12, 13, and 14 of gestation in the C57BL/6J mouse embryo. During this period intracellular cAMP levels and cAMP-dependent protein kinase (cAMP-dPK) levels in the palate transiently elevate. Cyclic AMP activates cAMP-dPK by binding primarily to two types of regulatory subunits of this enzyme, designated as RI and RII. To assess whether differential compartmentalization of the regulatory subunits occurs during palatal ontogeny, cytosolic, nuclear, and particulate fractions were prepared from day 12, 13, and 14 embryonic maxillary and palatal tissue. After photo-affinity labeling of each fraction with 8-azido [32P] cAMP, SDS-PAGE, and autoradiography, autoradiograms were analyzed densitometrically. The RI isoform predominated in the nuclear and particulate fractions on all three developmental days; whereas RII predominated in the cytosolic fractions. Thus, differential compartmentalization of cAMP-dPK may be a means by which cAMP dependent responses are regulated during palatogenesis.     

Changes in protein kinase activities during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat

Livers of rats fed the carcinogen 2-acetylaminofluorene (AAF) at a concentration of 0.025% were analyzed for protein kinase activities with [gamma 32P]ATP as substrate and either endogenous or exogenous (casein or histone) protein acceptors both in the presence or absence of cyclic nucleotides. Total protein kinase activity of the nuclear fraction, with exogenous histone or casein as substrate, was elevated during the first week of carcinogen administration. Total cytoplasmic kinase activities exhibited a pattern of activity change with maxima at about 25 and 42-49 days after the onset of carcinogen administration. Cyclic AMP levels rose steadily to approximately a 4-fold elevation by day 49 in livers of animals receiving carcinogen with the increase beginning prior to the development of externally visible nodular hyperplastic lesions. The findings demonstrate consistent and reproducible patterns of change in protein kinase activities that accompany AAF-induced hepatocarcinogenesis in the rat and provides the basis for a more detailed investigation of specific kinases.     

Postnatal ontogeny of uridine kinase in the cerebellum,hypothalamus, and cerebral cortex of the rat

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes inK _m with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.     

Subcellular alterations in cortisone treated rat lungs

Summary Lungs from cortisone treated rats were examined under the electron microscope. Major alterations appeared in the mitochondria; changes in the endoplasmatic reticulum and basement membranes were also observed. Isolated features noticed were extracellular and intracellular lattice structures, crystalloid formation, swelling of the Golgi apparatus and nuclear damage.This investigation was supported in part by an American Cancer Society Institutional Grant.     

Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells

With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction.