Total Protein Pool Research Articles

Cell Surface Glycoproteins Undergo Postbiosynthetic Modification of Their N-Glycans by Stepwise Demannosylation

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.

Identification and Quantitation of the Fatty Acids Composing the CoA Ester Pool of Bovine Retina, Heart, and Liver

Several proteins found in retinal photoreceptor cells (guanylate cyclase activating protein, protein kinase A, recoverin, and transducin) are N-terminally modified with the fatty acids 12:0, 14:0, 14:1n-9, and 14:2n-6, whereas similar proteins in other tissues contain only 14:0. It has been hypothesized that the acyl-CoA pool of the retina contains amounts of 12:0, 14:1n-9, and 14:2n-6 elevated over 14:0, in comparison to other tissues, and this accounts for the specificity of N-terminal fatty acylation. To test this hypothesis, we performed fatty acid analysis on total acyl-CoAs purified from bovine retina (light-adapted), heart, and liver. We also examined the N- and S-linked fatty acid composition of the total protein pools from these tissues. Acyl-CoAs were prepared from heart, liver, and retina and separated by high performance liquid chromatography (HPLC). Identities of peaks were based on HPLC of standard 12:0, 14:0, 14:1n-9, and 14:2n-6 CoAs. Total protein was subjected to base hydrolysis followed by acidic methanolysis to release S- and N-linked fatty acids, respectively, and fatty acid phenacyl esters were prepared for HPLC analysis. Retina had levels of 12:0 (2.7 +/- 2.1%), 14:1n-9 (2.9 +/- 2.2%), and 14:2n-6 (1.6 +/- 0.7%) CoAs below that of 14:0 CoA (7.0 +/- 1.8%). Likewise, heart levels of 14:2n-6 CoA (3.7 +/- 0.1%) were near and 12:0 (2.6 +/- 0. 6%) and 14:1n-9 (0.7 +/- 0.3%) CoAs were below that of 14:0 CoA (3.8 +/- 1.0%). Liver had levels of 12:0 (16.1 +/- 5.7%) and 14:2n-6 (8.1 +/- 1.2%) CoAs above and 14:1n-9 CoA (1.2 +/- 0.6%) below that of 14:0 CoA (5.9 +/- 0.8%). Fatty acid analysis of total protein showed that all tissues contained S-linked 16:0, 18:0, and 18:1n-9. Retina proteins contained N-linked 14:0, 14:1n-9, and 14:2n-6, whereas heart and liver had only 14:0. Our findings do not support the hypothesis that the CoA ester pool of the retina is enriched with 12:0, 14:1n-9, and 14:2n-6 over 14:0, in comparison to other tissues. This suggests that alternative models must be considered for the regulation of N-terminal fatty acylation of proteins in photoreceptor cells.

Affinity Biotinylation: Nonradioactive Method for Specific Selection and Labeling of Cellular Proteins

Biotinylation has become a popular alternative to radioiodination for labeling cell surface proteins, whereas labeling of the total cellular protein pool is usually achieved metabolically with [35S]methionine and [35S]cysteine. In this paper we describe a new technique in which total cellular lysate proteins that have been affinity bound to a solid phase are labeled efficiently with biotin. This labeling technique is preferable to direct biotinylation of cell lysate since the unreacted biotin can be readily removed from the sample by washing. The affinity step permits preselection of the molecules to be labeled, thereby decreasing the potential for nonspecific binding during subsequent immunoprecipitation. We applied this affinity biotinylation method to a human cellular lysate in order to preselect the total glycoprotein pool for subsequent immunoprecipitation of HLA class I. Following immunoprecipitation, SDS–PAGE, and Western blot, the biotinylated protein could be readily revealed by enhanced chemiluminescence. The results were comparable to those obtained by radiometabolic labeling and Western blot using a monoclonal antibody probe. Overall, the affinity biotinylation method is faster and more practical than conventional radiolabeling, without any loss in sensitivity.

Impact of alpha-skeletal actin but not alpha-cardiac actin on myoblast morphology.

Muscle differentiation involves a profound change in cell cytoarchitecture. This is accompanied by extensive isoform replacement in which the major non-muscle isoforms of the actin filament system are replaced by their muscle isoform counterparts. We have tested whether the sequential expression of the actin isoforms is functionally significant by precociously expressing the two striated muscle actins (alpha-skeletal and alpha-cardiac) in mouse myoblasts. The human alpha-skeletal and alpha-cardiac actin genes were transfected into mouse C2 myoblasts and clones expressing the human genes at the highest level were identified. Expression of the human alpha-skeletal actin gene was low with the highest mRNA level found to be 4% of that in adult human skeletal muscle. Clones expressing alpha-cardiac actin accumulated the mRNA up to 13% of the level of alpha-skeletal actin in adult human skeletal muscle. Despite the low level of alpha-skeletal actin expression, myoblasts transfected with this gene displayed a profound decrease in cell spreading. In contrast, alpha-cardiac actin had no impact on cell spreading. Neither alpha-skeletal actin nor alpha-cardiac actin had any impact on the total actin protein pool nor on the levels of the high molecular weight tropomyosins. The organisation of actin and tropomyosin into stress fibres was similar between transfected and control cells. We conclude that precocious expression of alpha-skeletal actin, but not alpha-cardiac actin, compromises myoblast morphology but not the ability of the cell to assemble stress-fibre-like structures.

Effects of brefeldin A on aggrecan core protein synthesis and maturation in rat chondrosarcoma cells.

In this paper, the effects of the fungal metabolite, brefeldin A, on the synthesis and maturation of aggrecan core protein precursor were studied in rat chondrosarcoma chondrocytes. The aggrecan core protein precursor was partially identified in total protein pools isolated from cell extracts based on its selective cleavage at a single site by the restriction protease factor Xa. During a 2-h labeling period with [3H]serine as precursor, brefeldin A inhibited the synthesis of mature aggrecan from its aggrecan core protein precursor consistent with an inhibition of chondroitin sulfate chain elongation and sulfation as described in the accompanying paper (Calabro, A., and Hascall, V. C. (1994) J. Biol Chem. 269, 22764-22770). This inhibition is presumably the result of the disruption of vesicular transport by brefeldin A, which isolates the aggrecan core protein precursor at the level of the trans-Golgi cisternae from the enzymes for chondroitin sulfate chain elongation and sulfation located in the trans-Golgi network. Brefeldin A also inhibited the exocytosis of all radiolabeled secretory proteins from the cell layer into the medium compartment, which is also consistent with the disruption of vesicular transport attributed to this metabolite. Although total protein synthesis was inhibited by 12% in the presence of brefeldin A, the aggrecan core protein precursor accumulated within the cell layer indicating that the inhibition of chondroitin sulfate synthesis by brefeldin A was not the result of a lack of aggrecan core protein precursor. When the brefeldin A block was removed and cultures chased in the presence of cycloheximide to prevent new protein synthesis, vesicular transport through the cell was re-established and chondroitin sulfate chains were added to a large proportion of the aggrecan core protein precursor that had accumulated during the brefeldin A block. These results suggest that the machinery for chondroitin sulfate synthesis and for protein exocytosis, that were disrupted by brefeldin A treatment, recover after removal of the brefeldin A, even in the presence of cycloheximide, and that the structures involved in these processes reassemble from previously existing proteins. Interestingly, two other proteins with the same relative abundance as the aggrecan core protein precursor were observed. An approximately 210-kDa protein with the characteristics of the fibronectin subunit, and an unidentified approximately 150-kDa protein which was efficiently cleaved by the protease Xa enzyme.

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes.

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

Lipophilic soluble protein represents labile protein in rat liver.

Lipophilic proteins, having higher turnover rates than the average of the total soluble protein pool, were separated from soluble rat-liver proteins on alkyl agarose column. The quantitative immunoprecipitation, using the antibody against the lipophilic proteins, showed that the soluble proteins from the liver of fed rats contained about twice as much lipophilic proteins as that of 3-day-starved rats. This result indicates that the lipophilic proteins, at least in part, represent labile protein in rat liver.

Age-related changes in total protein and collagen metabolism in rat liver

Liver collagen levels are determined by a balance between synthesis and degradation, processes known to have rapid rates in growing animals. We report age-related changes in liver collagen synthesis and degradation rates, as well as protein synthesis rates, in rats at five ages from 1 to 24 mo. Fractional collagen synthesis rates were determined after injection of [14C]proline with a flooding dose of unlabeled proline and its incorporation as hydroxy-[14C]proline into proteins. Fractional protein synthesis rates were based on the uptake of [14C]proline into proteins. Fractional collagen degradation rates were calculated from the difference between collagen fractional synthesis and deposition rates. Fractional rates of collagen synthesis were similar between 1 mo (23.0% ± 4.6%/day) and 24 mo (19.6% ± 3.4%/day) of age. Collagen deposition into the extracellular matrix was extremely low at every age studied; therefore degradation pathways accounted for the bulk of the collagen synthesized. The mean fractional synthesis rate for the total protein pool was unaltered between 1 mo (105.0% ± 7.2%/day) and 15 mo (89.9% ± 6.0%/day) of age, after which it increased to 234.9% ± 33.0%/day (p < 0.05) by 24 mo of age. These results indicate that liver collagen and total protein synthesis rates were maintained at relatively high levels during development and maturity but that protein synthesis rates were highest in senescent animals. (HEPATOLOGY 1991:14:1224–1229.)

Age-related changes in total protein and collagen metabolism in rat liver

Liver collagen levels are determined by a balance between synthesis and degradation, processes known to have rapid rates in growing animals. We report age-related changes in liver collagen synthesis and degradation rates, as well as protein synthesis rates, in rats at five ages from 1 to 24 mo. Fractional collagen synthesis rates were determined after injection of [14C]proline with a flooding dose of unlabeled proline and its incorporation as hydroxy-[14C]proline into proteins. Fractional protein synthesis rates were based on the uptake of [14C]proline into proteins. Fractional collagen degradation rates were calculated from the difference between collagen fractional synthesis and deposition rates. Fractional rates of collagen synthesis were similar between 1 mo (23.0% +/- 4.6%/day) and 24 mo (19.6% +/- 3.4%/day) of age. Collagen deposition into the extracellular matrix was extremely low at every age studied; therefore degradation pathways accounted for the bulk of the collagen synthesized. The mean fractional synthesis rate for the total protein pool was unaltered between 1 mo (105.0% +/- 7.2%/day) and 15 mo (89.9% +/- 6.0%/day) of age, after which it increased to 234.9% +/- 33.0%/day (p less than 0.05) by 24 mo of age. These results indicate that liver collagen and total protein synthesis rates were maintained at relatively high levels during development and maturity but that protein synthesis rates were highest in senescent animals.

Synthesis and Accumulation of Protein and Carbohydrases Along the Rat Villus Column

Summary: Enterocytes of the intestinal mucosa of infant and adult rats continuously proliferate in the crypt, mature as they migrate along the villus column, and are discharged from the villus tip. We examined the synthesis patterns of total protein, lactase‐phlorizin hydrolase, sucrase‐isomaltase, and maltase‐glucoamylase as well as the accumulation of these enzymes in cells during migration along the villus. Labeled leucine was administered intraperitoneally to suckling and young adult rats, and radioactivity was determined in protein and digestive carbohydrase pools of developing villus cells separated sequentially from tip to base of the villus column. The developing cells were found to continuously accumulate protein and carbohydrases as they ascended the villus column. In addition, incorporation of radioactivity into total protein and carbohydrase pools occurred at generally constant rates along the length of the villus. These studies showed that the differentiated enterocyte of both infant and young adult rat intestine exhibits a pattern of continuous growth while migrating the length of the villus column and maintains synthesis of protein and digestive carbohydrases at generally constant rates during this time.

Synthesis and Accumulation of Protein and Carbohydrases Along the Rat Villus Column

Enterocytes of the intestinal mucosa of infant and adult rats continuously proliferate in the crypt, mature as they migrate along the villus column, and are discharged from the villus tip. We examined the synthesis patterns of total protein, lactase-phlorizin hydrolase, sucrase-isomaltase, and maltase-glucoamylase as well as the accumulation of these enzymes in cells during migration along the villus. Labeled leucine was administered intraperitoneally to suckling and young adult rats, and radioactivity was determined in protein and digestive carbohydrase pools of developing villus cells separated sequentially from tip to base of the villus column. The developing cells were found to continuously accumulate protein and carbohydrates as they ascended the villus column. In addition, incorporation of radioactivity into total protein and carbohydrase pools occurred at generally constant rates along the length of the villus. These studies showed that the differentiated enterocyte of both infant and young adult rat intestine exhibits a pattern of continuous growth while migrating the length of the villus column and maintains synthesis of protein and digestive carbohydrates at generally constant rates during this time.

Myristoylation of heparan sulfate proteoglycan and proteins occurs post-translationally in human colon carcinoma cells

We have recently shown that the heparan sulfate proteoglycan of human colon carcinoma cells is acylated with both myristate and palmitate, two long-chain saturated fatty acids. In this study we show that cycloheximide did not significantly inhibit the incorporation of myristic acid into either proteoglycan or total protein pool. This lack of inhibition occurred under a condition in which protein synthesis was inhibited > 90%. Cycloheximide, on the other hand, did not affect the incorporation of [ 3H]myristic acid into fatty acid nor the intracellular interconversion of myristate to palmitate. Characterization of fatty acyl moiety in the proteoglycan and protein by reverse-phase HPLC revealed that ∼ 60% of the covalently bound fatty acids was myristate and the remaining 40% was palmitate. These results indicate that in human colon carcinoma cells myristoylation of heparan sulfate proteoglycan and proteins occurs post-translationally, presumably in the Golgi complex.

Fodrin degradation by calcium-activated neutral proteinase (CANP) in retinal ganglion cell neurons and optic glia: preferential localization of CANP activities in neurons

The activity of calcium-activated neutral proteinases (CANPs) toward endogenous substrates was measured in axons of retinal ganglion cell (RGC) neurons and separately in adjacent optic glia under in vitro conditions that preserved the ultrastructure and anatomic relationships between these cellular elements. RGC neurons and optic glia both expressed CANP activity. In contrast to RGC axons, which contained at least two CANP activities with calcium requirements in the millimolar (CANP A) and micromolar (CANP B) range (Nixon et al., 1985), CANP activity in optic glia was detectable only at millimolar calcium concentrations. When maximally activated, CANP(s) in optic glia exhibited a broad specificity for endogenous proteins but degraded larger proteins at a faster rate. The cytoskeletal protein fodrin (brain spectrin) was among the most susceptible endogenous substrates in RGC axons or glia. The similar properties of fodrin in neurons and glia, including its susceptibility to a purified millimolar calcium-sensitive brain CANP (mCANP), provided the basis for using this protein as a substrate to compare the relative activity of neuronal and glial CANPs in situ. Fodrin degradation mediated by CANPs proceeded at least 6 X more rapidly in intact RGC axons than in optic glia. Comparable differences in the relative degradation rates of the total neuronal and glial protein pools were also observed. These results indicate that the potential activity of CANPs is substantially greater in RGC neurons than in glia. The enormous potential activity and preferential localization of multiple CANP activities in RGC neurons support previously hypothesized roles for CANPs in the processing of axonally transported proteins and in the regulation of neuronal cytoskeletal dynamics and geometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP.

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.

Inhibition of cardiac proteolysis by colchicine. Selective effects on degradation of protein subclasses.

1. The effect of colchicine (2.5 microM) on cardiac protein turnover was tested with foetal mouse hearts in organ culture. 2. Colchicine had no effect on protein synthesis, but inhibited total protein degradation by 12-18%. Lumicolchicine, which lacks colchicine's ability to disaggregate microtubules, but shares its non-specific effects, did not alter protein degradation. 3. The colchicine-induced inhibition of protein degradation was accompanied by significant changes in cardiac lysosomal enzyme activities and distribution. 4. Colchicine inhibited the degradation of organellar proteins, including mitochondrial cytochromes, more than that of cytosolic proteins. 5. Colchicine decreased the rate of myosin degradation and the rate of proteolysis of the total protein pool to a similar extent. Since the regulation of myosin degradation does not involve lysosomes, this suggests that colchicine affects non-lysosomal as well as lysosomal pathways. 6. Release of branched-chain amino acids from colchicine-treated hearts was disproportionately decreased, suggesting that colchicine increased their metabolism. 7. It is concluded that colchicine, via its actions on microtubules, exerts important inhibitory effects on cardiac proteolysis. Colchicine is especially inhibitory to the degradation of organellar proteins, including mitochondrial cytochromes. Its inhibitory effects may be mediated in part via lysosomal mechanisms, but non-lysosomal mechanisms are probably involved as well.

The rate of degradation of liver proteins

In adult young rats, administration of large doses of glucocorticoid completely arrested the increase in the amount of total liver protein, but induced alanine aminotransferase to the maximum level. The rates of degradation of the total protein pool and of the alanine aminotransferase pool were estimated by measuring the rates of disappearance of radioactivity from the pools which had been prelabeled with radioactive amino acids. The apparent half-life values of total protein were 6.1 days in normal livers and 3.2 days in livers receiving 10 mg of steroid per day. The apparent half-life value of the enzyme was 3.3 days in normal livers, and in glucocorticoid-treated livers the values were 10.2 days when the enzyme was labeled before induction and 2.4 days when the enzyme was labeled after the tissue had reached the maximally induced enzyme level. These apparent half-life values correlated closely with the changes in the size of each pool. The decay rates of radioactive proteins obtained from livers in which the size of pool was maintained constant by the glucocorticoid reflect the true rate of degradation of each macromolecular species.

Studies on the morphopoiesis of the head of phage T-even. V. The formation of polyheads.

The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T4 amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.

Environmental control of enzyme synthesis and degradation

Glucocorticoid administration produces an increase in precursor incorporation into rat liver glutamic-alanine transaminase in the whole animal and in liver preparations incubated in vitro. The augmented incorporating ability was present in both the microsomal and cell sap fractions. The effect both in vivo and in vitro was specific, with little or no increase observed in the total soluble protein pool of the cell. From these results, together with information obtained from measurements of the biological half-life of the enzyme, it is concluded that the increase in tissue level of the enzyme consequent to glucocorticoid administration results from an increased rate of biosynthesis. The possible role of environmental control of enzyme levels in cells through effects on degradation rates is discussed. The increased rate of precursor incorporation into liver glutamic-alanine transaminase in vivo was aleady at a maximum value 12 hours after initiation of hormone administration, in contrast to the enzyme level which had not increased by 12 hours and reached a peak only after 5 days. These findings suggest the possibility that there is a simultaneous induction by glucocorticoids of all the liver enzymes responsive to the hormone.