Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.     

A substance in L-929 cell extracts which replaces the ascorbate requirement for prolyl hydroxylase in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and the level of prolyl hydroxylase activity in these cells

Reductant used as cofactor for the prolyl hydroxylase reaction, was measured by a tritium release assay modified from an enzyme assay by making all components of the assay system saturating except for the reductant, but including prolyl hydroxylase. Reduced glutathione (6 mm), which had little activity as a cofactor, and thymol (0.1 mm), an antioxidant which exhibited no cofactor activity at all, were required for optimal proline hydroxylation dependent on reducing cofactor, with thymol fulfilling the previously described requirement for catalase. Ascorbate, cysteine and 6,7-dimethyltetrahydropterin were active as cofactors, in descending order of activity at equimolar concentrations, and activity was concentration dependent for all of these compounds. Sonicates of stationary phase L-929 cells which exhibit ascorbate-independent proline hydroxylation in culture contained reducing cofactor which could replace ascorbate in the cofactor assay, while sonicates of log phase cells which exhibit an ascorbate requirement in culture contained about one-third or less of that amount. NADH and NADPH, which themselves have little or no activity as cofactor, increased the cofactor activity of log phase cell sonicates but had relatively little effect on the activity of stationary cell sonicates suggesting that the cofactor is in a more reduced state in stationary phase. Within 24 h after replating dense, stationary phase cell cultures at low density, conditions where cells return to ascorbate dependence, prolyl hydroxylase activity had decreased to one-fifth the original activity while the concentration of functional reducing cofactor had decreased to less than 1% of its original concentration, largely as a result of oxidation. Ascorbate was not present in L-929 cells sonicates and the levels of tetrahydropterin and cysteine in sonicates could not account for the amount of cofactor activity exhibited by the sonicates in the assay system. Treatment of L-929 cultures with aminopterin did not decrease ascorbate independence, suggesting that tetrahydrofolate did not contribute significantly to cellular proline hydroxylation. These results suggest that an unidentified reductant present in L-929 cells can account for ascorbate-independent proline hydroxylation and also regulate prolyl hydroxylase activity in these cells and that cellular levels of reduced pyridine nucleotides may regulate the reduction state of this substance.     

Localization of collagen prolyl hydroxylase to the hepatocyte : Studies in primary monolayer cultures of parenchymal cells from adult rat liver

Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocytes prepared from normal or regenerated adult rat liver and primary non-proliferating monolayer cultures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47% of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore, prolyl hydroxylase was localized by immunofluorescence uniformly to the hepatocytes in culture. Cultured hepatocytes converted [¹⁴C]proline to [¹⁴C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.     

The effect of hypoxia on collagen synthesis in cultured 3T6 fibroblasts and its relationship to the mode of action of ascorbate.

When exposed to low oxygen tension, in the absence of added ascorbic acid 3T6 mouse fibroblast cultures in late log phase respond by increased lactate production and increased hydroxylation of proline in nascent collagen, which is paralleled by an increase in prolyl hydroxylase activity. After 6 h recovery from the anoxic stimulus, however, cultures still yield more prolyl hydroxylase than controls, but the effect on hydroxylation of nascent collagen has disappeared. These observations help to dissect the dual role of ascorbate which can stimulate hydroxylation both by increasing the amount of active enzyme and by a cofactor-like role; in addition, these observations may be relevant to wound healing.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

Studies on the mechanism of reduction of prolyl hydroxylase activity by D,L-3,4 dehydroproline.

When chick frontal bone cells in culture were exposed to d,l-3,4 dehydroproline, the specific activity of prolyl hydroxylase was markedly reduced, but the concentration of the protein antigenically related to prolyl hydroxylase was not decreased. The specific activity of purified prolyl hydroxylase from cells grown in d,l-3,4 dehydroproline was significantly lower than that of control cells. Preincubation of a homogeneous preparation of chick embryo prolyl hydroxylase with collagenous peptides containing [¹⁴C]d,l-3,4 dehydroproline resulted in a time-dependent decrease in the enzymatic activity. These observations suggest that the in vivo reduction in prolyl hydroxylase activity by dehydroproline could be either due to an interaction of the enzyme with collagenous peptides containing dehydroproline and/or the synthesis of an aberrant form of prolyl hydroxylase with decreased enzymatic activity.     

Suppression of fibroblast proliferation and lysyl hydroxylase activity by minoxidil

The effect of minoxidil on lysyl hydroxylase activity and proliferation of human skin fibroblasts in culture was examined. Exposure of cells to minoxidil resulted in a specific loss of lysyl hydroxylase activity, the extent of which was dependent on the concentration of minoxidil from 25 to 500 microM and the duration of the treatment from 6 to 48 h. This phenomenon was unaffected by culture conditions, i.e. ascorbic acid status, serum concentration, and cell density. Minoxidil added directly to cell extracts had no effect on lysyl hydroxylase activity, showing a requirement for intact cells. Mixing experiments with extracts of minoxidil-treated cells and controls gave additive results which rule out the possibility that a metabolite derived from minoxidil could be inhibiting the enzyme activity. The effect of minoxidil on fibroblast lysyl hydroxylase activity disappeared in the presence of cycloheximide, an inhibitor of protein synthesis. Moreover, the recovery of the enzyme activity that occurred after removal of minoxidil from the culture medium could be prevented by actinomycin D, an inhibitor of RNA synthesis. These results indicate that minoxidil may inhibit the synthesis of lysyl hydroxylase in the cell. In addition to suppressing fibroblast lysyl hydroxylase activity, minoxidil caused inhibition of cell growth within 48 h in a manner dependent on the concentration from 10 to 1000 microM, the latter resulting in almost complete cessation of cell proliferation. This effect was not accompanied by cytotoxicity as judged by the criteria of dye exclusion, plating efficiency, growth recovery, and protein synthesis. The inhibition of fibroblast proliferation by minoxidil appeared to be related to its ability to inhibit DNA synthesis measured by incorporation of tritiated thymidine into acid-precipitable material.     

Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation of prolyl hydroxyla.

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 mug of enzyme protein per 10(8) cells and 40-50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein but only 15-20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and incultured tendon cells had the same apparent size and the same activity per mug of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme. When freshly isolated cells were incubated for 2 h in the presence of 40 mug per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 mug per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not icrease the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve "activation" of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

Hydralazine differentially increases mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase whereas it decreases pro alpha 1(I) collagen mRNAs in human skin fibroblasts.

We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the alpha and beta subunits of prolyl 4-hydroxylase (PH), a key post-translational modifying enzyme in collagen biosynthesis. Hydralazine exerts a paradoxical effect on collagen biosynthesis in cultured fibroblasts. Cells exposed to hydralazine synthesize substantially reduced amounts of collagen, which is severely deficient in hydroxyproline. Surprisingly, however, the level of prolyl hydroxylase activity assayed in extracts of treated cells is markedly increased, suggesting overproduction of the enzyme. Hybridization analysis indicated that in untreated cells the concentration of the alpha PH subunit mRNA was about 20-25% of the beta PH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both alpha and beta subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The alpha subunit mRNA was maximally increased within 24 h, whereas the beta subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for pro alpha 1(I) collagen were virtually eliminated by 72 h. This study demonstrates that the increased prolyl hydroxylase activity is a direct result of hydralazine-mediated increases in steady state mRNA content for the alpha and beta subunits of this enzyme. Moreover, the earlier induction of alpha PH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the alpha subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for pro alpha 1(I) collagen.     

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.     

Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21-C13 cells by the addition of serum

BHK21/C13 cells placed in medium containing low (1%) serum ceased DNA synthesis within 4 days. DNA synthesis recommenced 10 h after the readdition of serum (to 10%) to cells incubated for 6 days in serum-depleted medium. Two peaks of thymidine incorporation were observed at 12–13 h and 15–17 h, followed by a single peak of dividing cells at 25 h. The two peaks of incorporation represent variation in the extent of DNA replication during a single synchronous S phase.Uridine, deoxyadenosine and deoxyguanosine kinase activities did not decline in serum-depleted cells and, after the addition of serum, their activities showed cyclical variation about a mean involving two-fold changes in enzyme specific activity. All other enzyme activities examined were markedly decreased in resting cells.Ornithine decarboxylase activity increased 15-fold within 5 h of serum addition, but had returned to the resting level by 8 h. There was no apparent correlation between this alteration of enzyme activity and the rate of RNA synthesis.DNA polymerase, thymidine kinase and deoxycytidine kinase activities all decreased further within 4 h of the addition of serum, followed by several-fold increases in activity. The peak of DNA polymerase activity corresponded to, and encompassed, both peaks of DNA synthesis. However, thymidine and deoxycytidine kinase activities, although exhibiting two activity maxima corresponding to the peaks of DNA synthesis, were at their highest levels in G2.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.     

Regulation of collagen post-translational modification in transformed human and chick-embryo cells.

Changes in the regulation of collagen post-translational modification in transformed cells were studied in three established human sarcoma cell lines and in chick-embryo fibroblasts freshly transformed by Rous sarcoma virus. The collagens synthesized by all but one of these and by all the control human and chick-embryo cell lines were almost exclusively of types I and/or III. The relative rate of collagen synthesis and the amounts of prolyl hydroxylase activity and immunoreactive protein were markedly low in all the transformed human cell lines. The other enzymes studied, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase, never showed as large a decrease in activity as did prolyl hydroxylase, suggesting a more efficient regulation of the last enzyme than of the three others. The chick-embryo fibroblasts freshly transformed by Rous sarcoma virus differed from the human sarcoma cells in that prolyl hydroxylase activity was distinctly increased, whereas the decreases in immunoreactive prolyl hydroxylase protein and the three other enzyme activities were very similar to those in the simian-virus-40-transformed human fibroblasts. It seems possible that this increased prolyl hydroxylase activity is only a temporary phenomenon occurring shortly after the transformation, and may be followed by a decrease in activity later. The newly synthesized collagens of all the transformed cells that produced almost exclusively collagen types I and/or III had high extents of lysyl hydroxylation, and there was also an increase in the ratio of glycosylated to non-glycosylated hydroxylysine. The data suggest that one critical factor affecting modification is the rate of collagen synthesis, which affects the ratio of enzyme to substrate in the cell.     

Selective changes in tRNA methyltransferase activity in confluent monolayers of WI-38 cells stimulated to proliferate.

In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzyme show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2 microgram/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethyl-guanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.     

Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.