Turnover of plasma membrane proteins in rat hepatoma cells and primary cultures of rat hepatocytes

The half-lives of turnover of plasma membrane proteins in rat hepatoma tissue, culture cells, and in primary cultures of rat hepatocytes have been analyzed after resolution by two-dimensional gel electrophoresis. Cell membranes were externally labeled via iodination catalyzed by lactoperoxidase and glucose oxidase. A bimodal pattern of turnover was found for the externally oriented plasma membrane proteins of rat hepatoma cells. Three glycoproteins analyzed in these cells had an average t 1/2 of 22 h while eight proteins which did not bind to concanavalin A had an average t 1/2 of 80 h. In contrast, more heterogeneous rates of turnover were found for the externally oriented plasma membrane proteins of primary cultures of hepatocytes. Most, if not all, of the membrane proteins accessible to iodination in these cells were glycoproteins. Among the glycoproteins resolved by two-dimensional polyacrylamide electrophoresis, the receptors for asialoglycoproteins had the shortest half-lives (18 h). Other glycoproteins, mostly with higher molecular weights and different isoelectric points, showed a spectrum of half-lives ranging from 16 to 99 h. The turnover rates of membrane proteins of primary cultures of rat hepatocytes were also determined with [3H]- and [35S]methionine labeling of cells. Heterogeneous rates of turnover again were found among the labeled glycoproteins and nonglycoproteins. Among the 10 glycoproteins individually analyzed, the half-lives range from 17 to 67 h. Among the 21 proteins which do not bind to concanavalin A, the half-lives range from 18 h to more than 100 h. Three proteins analyzed showed an apparent biphasic pattern of turnover, having a fast phase with a half-life of 4-6 h and a slow phase with a half-life of 15-29 h. Several nonglycoproteins, including clathrin and actin associated with membrane vesicles had extremely long half-lives. The more than 5-fold difference in the half-life between clathrin and the receptors for asialoglycoproteins, which coexist in coated pits indicates that intrinsic proteins of the coated pits turn over at a different rate than peripheral components.     

Changes in RNA in relation to growth of the fibroblast: II. The lifetime of mRNA,rRNA, and tRNA in resting and growing cells

The stabilities of the principal classes of RNA have been studied in resting and exponentially growing mouse fibroblast lines 3T6 and 3T3. Cytoplasmic mRNA, labeled with tritiated uridine and isolated by virtue of its poly A content, is equally stable in resting and growing cells, displaying a half-life of about 9 hr. We conclude that the accumulation of poly A(+) mRNA during transition from resting to growing state is due not to an increase in its stability, but to an increase in its rate of formation.The stability of cytoplasmic rRNA was measured after labeling with ³H-methyl-methionine. In agreement with the results of previous studies, we found that rRNA is stable in growing cells and unstable in resting cells. Quite unexpectedly, the 18S and 28S rRNA of resting cultures were found to differ appreciably in turnover rate. In both 3T6 and 3T3, the half-life of 28S RNA is about 50 hr, and that of 18S RNA about 72 hr. For this reason, though growing cells should synthesize the two ribosomal subunits in equal numbers, resting cells should synthesize more of the larger subunits than of the smaller. tRNA is unstable under all conditions. Its half-life is 36 hr in resting cells and about 60 hr in growing cells.     

Regional differences in the turnover of neuronal histamine in the rat brain

The turnover rate of histamine (HA) and the half-life of neuronal HA were estimated in 9 regions of the rat brain following pargyline-induced accumulation of tele-methylhistamine (t-MH). The turnover rate was the highest in the hypothalamus (108.7 ng/g/hr). The striatum also showed a high turnover rate (80.2 ng/g/hr) despite much lower levels of HA and t-MH, as compared with the levels in the hypothalamus. The turnover rate was relatively high in the thalamus, cerebral cortex, amygdala and midbrain, but it was very low in the cerebellum. t-MH accumulation in the spinal cord was nil. The HA levels were reduced to various degrees (from nil to less than 40% of the control) by (S)-alpha-fluoromethylhistidine, depending on the regions studied. The neuronal HA content of each brain region was subsequently estimated, and the half-life of neuronal HA in each region was calculated. The half-life of neuronal HA was the shortest (7.7 min) in the striatum, while it was long (about 50 min) in the hypothalamus and thalamus. Half-life values of about 20 min were obtained in other regions. These results show the high levels of histaminergic activity in some parts of the telencephalon, thalamus and midbrain as well as the hypothalamus.     

Microbial Assimilation of Hydrocarbons: Phospholipid Metabolism

An analysis of the turnover of the major phospholipids of Micrococcus cerificans growing or nongrowing cultures. The turnover rates of (14)C-PE and (14)C-PE were 61.5% of the total phospholipid, exhibited no significant rate of turnover in either growing or nongrowing cultures. The turnover rates of PE-(14)C and PE-(32)P were 3.2% per hr and 1.2% per hr, respectively. Phosphatidylglycerol (PG) exhibited a turnover rate of 11% and 7.7% per hr for (14)C and (32)P, respectively, indicating an extremely slow metabolism. PG metabolism was examined in greater detail, and the data indicated a preferential 75% incorporation of glycerol-1,3-(14)C into the unacylated portion of the PG molecule. The turnover of cardiolipin (CL) was extremely slow in growing cells whereas nongrowing cells exhibited a 30% and 36% increase per hr for (14)C-Cl and (14)C-CL, respectively. Glycerol-1,3-(14)C was not converted to phospholipid fatty acid carbon; all radioactivity appeared only in the water-soluble backbone of the phospholipids. The kinetics of assimilation of hexadecane-1-(14)C into cellular lipids is presented. Radioactivity in neutral lipid increased approximately sevenfold over the growth cycle, whereas radioactivity in phospholipid increased 50-fold during the same time period. The incorporation of radioactive fatty acids derived from the direct oxidation of hexadecane-1-(14)C demonstrated differential kinetics of assimilation into PE, PG, and CL. The results indicated a rapid turnover of phospholipid fatty acids in M. cerificans growing at the expense of hexadecane.     

Turnover of newly synthesized cytochromes P-450scc and P-45011 beta and adrenodoxin in bovine adrenocortical cells in monolayer culture: effect of adrenocorticotropin

The turnover of newly synthesized cytochromes P-450scc and P-45011 beta, and adrenodoxin was investigated in bovine adrenocortical cells in primary monolayer cultures. Cells were pulse-radiolabeled with [35S]methionine, and specific newly synthesized enzymes were immunoisolated at various times following labeling and quantitated. Adrenocorticotropin (ACTH) treatment did not alter the average turnover rate of total cellular proteins or that of total mitochondrial proteins. The half-life of total cellular proteins of control and ACTH-treated cells was determined to be 20.5 and 23 h, respectively. The half-life of mitochondrial proteins of control and ACTH-treated cells was determined to be 42.5 and 44 h, respectively. The turnover rate of newly synthesized cytochrome P-450scc was approximately the same as total mitochondrial protein (t1/2 = 38 h), and was unchanged by ACTH treatment (t1/2 = 42 h). ACTH treatment did not greatly alter the turnover rate of adrenodoxin. The half-life of adrenodoxin from control and ACTH-treated cells was determined to be 20 and 17 h, respectively. However, ACTH treatment appeared to increase the half-life of cytochrome P-45011 beta from 16 h in control cells to 24 h in treated cells. The differential rate of turnover of mitochondrial proteins studied here supports the contention that mitochondria are subject to heterogeneous degradation. It appears that chronic treatment of bovine adrenocortical cells in culture with ACTH leads to increased steroidogenic capacity, primarily as a result of increased synthesis of steroidogenic enzymes, although, as shown for cytochrome P-45011 beta, ACTH action might also increase steroidogenic capacity by increasing the half-life of this steroid hydroxylase.     

Study on the turnover of the receptor for the third component of complement on human lymphoid cells.

The in vitro turnover of the receptor for the third component of complement (C3) was studied in normal peripheral blood lymphocytes (PBL) and in lymphoblastoid cells from established cell cultures of both "normal" and "malignant" origin. The turnover was evaluated by studying i) the disappearance rate of the C3-receptor in cells in which the protein synthesis was blocked by cycloheximide and puromycin, ii) the reexpression rate of the C3-receptor after treatment of the cells with either rabbit antiserum against B lymphocytes or mouse C activated through the alternative pathway by inulin. The results show that the C3-receptor of all the lymphoid cells has roughly a half-life of about 3 to 4 hr. However, the cultured lymphoblastoid cells were less sensitive than normal PBL to inhibition by cycloheximide and showed a faster reexpression rate of the C3-receptor. A spontaneous release of the receptor was found to occur, since a receptor-like activity was detected in the spent culture medium of long-term cultured lymphoid cells.     

Population kinetic study on the origin of guinea pig monocyte heterogeneity

Population kinetic studies were performed on guinea pig peripheral blood monocyte fractions isolated by counter-flow centrifugation elutriation following a single in vivo pulse of tritiated thymidine. Labeled large monocytes (volume 317 micron3; relative distribution 49%; circulating half-life 5.7 hr; and production rate 17,000 cells/ml blood/hr) accumulated in the circulation more rapidly, had a faster turnover time, and were produced in greater numbers than small monocytes (volume 283 micron3; relative distribution 34%; circulating half-life 10.8 hr; and production rate of 6100 cells/ml blood/hr). The kinetic data do not support a maturational sequence of small into large monocytes. Intermediate monocytes (volume 300 micron3; relative distribution 11%; circulating half-life 18.2 hr) and very large monocytes (volume 354 micron3; relative distribution 6%; circulating half-life 36.5 hr) had production rates, respectively, of only 1200 and 320 cells/ml blood/hr. Maxima in the labeling index curve for small and large monocytes suggested a generation time of 24 hr while grain count analysis revealed that these two cell fractions were derived from a precursor population with similar numbers of reductive divisions. Grain count analysis of intermediate and very large monocytes revealed that these cells differed from both small and large monocytes. Our data support the concept that monocyte subsets exist in guinea pig peripheral blood with different kinetics of production and survival.     

Metabolic properties of substrate-attached glycoproteins from normal and virus-transformed cells.

Balb/c 3T3, SV40-transformed 3T3 (SVT2), and Con A revertant variants of transformed cells leave a layer of glycoprotein on the culture substrate upon EGTA mediated removal of cells. The metabolic properties of this substrate-attached material (glycoprotein) have been examined. Pulse and cumulative radiolabeling experiments with glucosamine and leucine precursors established that this substrate-attached material accumulates on the substrate in growing cultures until cells have completely covered the substrate. The synthesis and/or deposition of the material diminished dramatically in cultures whose substrates had been completely covered with cells as observed microscopically, even though the contact-inhibited cell lines continued to make cell-associated and medium-secreted glycoproteins and transformed cells continued to divide and form multilayered cultures. Pulse-chase analysis using long periods of pulsing with radioactive leucine demonstrated that these glycoproteins are deposited directly on the substrate by cells and not subsequent to secretion into the medium. The substrate-attached material accumulated during long pulses was stably adherent to the substrate and displayed little appreciable turnover during 3 days of chasing of either sparse or dense cultures. Short-term pulse-chase analysis with leucine revealed two metabolically different pools of material-one which turns over very rapidly with a half-life of 2-3 hr (observed in both low-density and high-density cultures) and a second pool which is stably deposited on the substrate and whose proportion increased with the length of the radiolabeling period. No appreciable differences in the metabolic properties of substrate-attached material were observed in the three cell types studied during growth on a plastic substrate. These results are discussed with regard to the implicated roles of these glycoproteins in in mediating adhesion of normal and virus-transformed cells to the substrate.     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Activities of three lysosomal enzymes--acid RNase. N-acetyl-beta-D-glucosaminidase and acid phosphatase--were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to fivefold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown also increased as cultures approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH4Cl, but the rate of turnover in the presence of NH4Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops.     

Cytolytic antitumor effector cells in long-term cultures of human tumor-infiltrating lymphocytes in recombinant interleukin 2

Summary Lymphocytes infiltrating human solid tumors (TIL) and autologous peripheral blood lymphocytes (A-PBL) were cultured with 1000 units/ml of recombinant interleukin 2 (rIL2) in long-term cultures. TIL isolated from 26 primary squamous cell carcinomas of the head and neck expanded better (P<0.01) and achieved higher total lytic units of activity against fresh tumor cell targets (P<0.05) than A-PBL. TIL obtained from primary hepatocellular carcinomas (n=7) showed a higher degree of expansion than those from metastatic liver tumors (n=7). Further, TIL from metastatic tumors of the head and neck, liver, and ovary were delayed up to 50 days in their proliferative response to rIL2. Long-term mass cultures in rIL2 of TIL, A-PBL, or normal PBL were serially monitored for cytotoxicity with different cultured and fresh tumor cell targets and for phenotypic markers of the predominating cell populations. Antitumor cytotoxicity was found in cultures enriched in CD3 + Leu19 + and/or CD3-Leu19 + cells. Two-color sorting of such cultures followed by cytotoxicity assays confirmed that the human antitumor effectors expressed either the CD3 + Leu19 + or CD3-Leu19 + phenotype. CD3 + Leu19- cells had little or no antitumor cytotoxicity. The two types of Leu19 + effector cells were present in low numbers in fresh TIL, A-PBL, or normal PBL; in contrast, in some rIL2-expanded long-term cultures, they represented a majority of proliferating cells. This study identifies for the first time two types of antitumor effector cells in rIL2 cultures of human TIL, one of which may represent activated natural killer cells on the basis of the absence of the CD3 and expression of the Leu19 antigen. These antitumor effector cells mediate non-MHC-restricted cytotoxicity of fresh or cultured tumor cell targets of different histologic types.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

Renewed DNA synthesis in senescent WI-38 cells by expression of an inducible chimeric c-fos construct.

As normal human cells approach the end of their proliferative lifespan in vitro they lose responsiveness to a variety of growth factors, to which they respond with DNA replication when they are young. Recently it has been reported that the protooncogene c-fos is not expressed in senescent cells (Seshadri and Campisi, 1990). In this study we have found that both c-jun and jun B, partners of c-fos in heterodimeric transactivating complexes, are equivalently expressed in young and senescent cells at both early (1-6 hr) and late (12 or 16 hr) time points following serum stimulation of quiescent cells. We have also investigated the effect of the enforced expression of c-fos in senescent WI-38 cells using an inducible construct carrying the murine c-fos gene under the control of the sheep metallothionein promoter. We have found that the transient transfection and subsequent activation of the conditional promoter with Zn++ stimulated DNA synthesis in a significant fraction of senescent cells which had completed 90%-95% of their proliferative lifespan. However, populations which had completed 100% of their proliferative life span and nondividing cultures which had been selected with BrdU did not respond to the expression of the c-fos gene. These results demonstrate that one of the primary events associated with senescence in human cells is the suppression of c-fos gene expression, but additional phenotypic changes must also occur in order to explain the ultimate loss of proliferative responsiveness of these cells.     

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.     

A change in the stability of globin mRNA during the induction of murine erythroleukemia cells

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.     

Proliferation kinetics of plasma cells and of normal haemopoietic cells in multiple myeloma

DNA synthesis time (Ts) and 3H thymidine (TdR) labelling index (LI) of bone marrow (BM) myelomatous plasma cells (PC) and of the residual haemopoietic cell population (RHCP) were measured by in vitro quantitative 14C-TdR autoradiography in five patients with multiple myeloma (MM) in different phases of disease (three at presentation and two at relapse) and in one patient with solitary extra-osseous myeloma. One other patient with plasma cell leukaemia (PCL) was studied during an initial relapse phase and later during the leukaemic terminal phase. PC Ts was 18.8 +/- 3.7 (from 13.3 to 25.0) hr and PC LI was 2.5 +/- 1.8% (from 1.0 to 6.3%). In the case of PCL, circulating PC had a Ts of 14.4 hr and a LI of 3.1. From these experimental measurements, the fractional turnover rate (FTR-percentage of cells produced per unit time) and the potential doubling time (Td) of BMPC were calculated assuming that all BMPC were in a steady-state at the time of the study. BMPC FTR was 3.53 +/- 2.3% cells per day (from 1.2 to 6.72) and BMPC Td was 46.8 +/- 27.5 days (from 15.0 to 75.4). Comparison with results obtained in BM blasts of children with acute lymphoblastic leukaemia (ALL) indicated that BMPC had a lower proliferative activity (P less than 0.001), although BMPC Ts was not significantly different. In two patients a tumour doubling time of 6 and 13 months was determined by clinical follow up. Comparison of this parameter with Td showed a cell loss factor of more than 90% in both patients. Kinetic data relative to RHCP showed slight variations with respect to those found in normal subjects, with a general tendency towards a prolongation of Ts and a reduction of LI.     

Phosphatidylcholine and phosphatidylethanolamine metabolism during lens fiber cell formation

Phosphatidylinositol is metabolized with a half-life of about 5 h in lens epithelial cells of 6-day-old embryonic chickens. When these cells differentiate to form lens fiber cells, however, phosphatidylinositol turnover virtually ceases. The present study was undertaken to determine whether there is a similar change in the metabolism of phosphatidylcholine and phosphatidylethanolamine. [32P]Orthophosphate was injected into 6-day-old chicken embryos, and the incorporation of label into phosphatidylcholine and phosphatidylethanolamine was followed for 48 h. The specific activities of the precursors phosphorylcholine and phosphorylethanolamine were also measured during this time. The data were then analysed by means of a simple kinetic model to determine the rate of synthesis and the half-life of each phospholipid. The results showed that phosphatidylcholine is synthesized at a rate of about 1.2 X 10(-20) mol/s per cell in the lens epithelial cells, and 6.4 X 10(-20) mol/s per cell in the fiber cells. Phosphatidylethanolamine is synthesized at approximately 0.9 X 10(-2)) mol/s per cell in the epithelial cells, and 4.0 X 10(-20) mol/s per cell in the fiber cells. Both phospholipids are stable in both the epithelial cells and in the fiber cells, with half-lives of 48 h or greater. Thus, although phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol all experience an increase in synthesis following lens fiber formation, the previously observed decrease in phosphatidylinositol turnover accompanying differentiation is a specific effect.     

Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain

1. The turnover of cerebral histones and DNA after injection of [4,5-(3)H]leucine or [methyl-3-(3)H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.     

Regulation of Proenkephalin Synthesis in Adrenal Medullary Chromaffin Cells

The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin-derived peptides with trypsin and carboxy-peptidase B, and quantitation of radioactivity incorporated into Met-enkephalin following reversed-phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10-fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 microM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, or proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three- to eight-fold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half-life for turnover of Met-enkephalin sequences was 3-4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.