Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

The influence of exercise on muscle lysosomal enzymes

Representative lysosomal enzyme activities were measured in muscles taken 0 h and 24 h after an acute exercise run that was completed without any overt signs of fatigue. The animals had progressed 2 and 4 weeks into a standard exercise program which typically produces adaptive changes in the working muscles. There was an increase in acetylglucosaminidase activity (12%) in the fast-twitch red muscle section of all animals that participated in the training program. This small increase may be representative of a delayed response found after more exhausting exercise. The single exercise bout, however, did not cause any acute change in lysosomal activity nor alter the partition of lysosomal enzymes between the "free" and particulate fractions. Thus, altered lysosomal enzyme activity does not appear to be a contributing influence that challenges muscle fiber homeostasis during moderately intense running.     

水杨酸对丹参培养细胞中迷迭香酸生物合成及其相关酶的影响

迷迭香酸(RA)是丹参中一种重要的酚酸类次生代谢物。为探讨水杨酸(SA)诱导子对丹参悬浮培养细胞中RA的生物合成及其相关酶的影响,考察了SA诱导子和酪氨酸氨基转移酶(TAT)的竞争性抑制剂(AOPP)对RA合成积累量、苯丙氨酸解氨酶(PAL)和TAT活性的影响。发现在培养的第6天用浓度为6.25 mg/L的SA处理后,PAL活性在诱导后4 h出现高峰,为对照组水平的124%;RA的积累量在诱导后8 h出现峰值(5.914±0.296)mg/g。用浓度为0.1μmol/L的AOPP处理,6 h后AOPP对TAT活性影响较小(与对照组无显著差异),但明显抑制了PAL活性(为对照组水平的44%),且在PAL活性明显降低的同时RA的积累量显著减少(4.709±0.204)mg/g。进一步用0.1μmol/L AOPP和6.25 mg/L SA共处理,AOPP对PAL的抑制作用可得到一定程度的缓解,且RA的积累量较AOPP单独处理的高。表明SA可以诱导丹参悬浮培养细胞中RA积累量的增加,且在RA合成过程中PAL的限速作用比TAT明显。     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

Nonlatent and latent hepatic glutathione-insulin transhydrogenase activity during perinatal development and liver regeneration in rats and in rat placenta

We have studied glutathione-insulin transhydrogenase (GIT) activity in differentiating rat liver during parturition and neonatal growth and during compensatory liver growth. Parturition is characterized by a rapid but transient increase in total (i.e., nonlatent plus latent) hepatic GIT activity resulting from changes in the quantity (Vm) of the enzyme while neonatal growth is characterized by an increase in the nonlatent (active) form which persists until just prior to weaning. During liver regeneration following partial hepatectomy, GIT activity/mg protein is lowest after 12 h of regeneration and then progressively increases exceeding the control levels after 72 h of regeneration. Placenta from near-term rats contain a significant concentration of GIT which is immunologically similar to hepatic GIT.     

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.     

Galactose metabolism in regenerating rat liver.

1. Rats trained on a controlled lighting and feeding schedule were subjected to partial hepatectomy or sham operation. 2. After a large period of about 6h the activity of UDP-galactose 4-epimerase increased threefold, reaching a maximum 4 days after partial hepatectomy, and returned to normal values within a fortnight. 3. The enzyme pattern of the UDP-galactose-glycoprotein galactosyltransferase was biphasic, one peak appearing at 20 h, the second at 72 h after partial hepatectomy. 4. The rise in enzyme activities could be blocked by the injection of actinomycin D, and the Km values for UDP-glucose and UDP-galactose were nearly identical in regenerating and adult liver. It is therefore concluded that the increase in enzyme activity is due to synthesis de novo of enzyme protein.     

The activity of gamma-glutamyltranspeptidase in regenerating rat liver

gamma-Glutamyltranspeptidase is expressed at low levels in the liver of the male Fischer 344 rat where it exhibits 15-fold purification and 33% recovery in isolated plasma membranes. While the activity of the enzyme is unaltered in regenerating liver 24 h after partial hepatectomy, it increases steadily thereafter over a period of one week. Seven days after partial hepatectomy the enzyme is maximally activated: 5.6-fold in liver homogenates and 5.3-fold in isolated liver plasma membranes. The enzyme declines in activity over the next fourteen days and is expressed at normal levels three weeks after partial hepatectomy. These results demonstrate that the activity of gamma-glutamyltranspeptidase increases in regenerating liver but that the increase is out of phase with the proliferative response.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Polyamine metabolism in regenerating livers from normal and hypocalcemic rats

There is a marked increase in the concentration of putrescine during the first ten hours following partial hepatectomy in rats. The concentration of spermidine also increases but to a smaller degree. Putrescine levels return to normal between 10 and 24 hours after the operation, whereas the increased spermidine level is maintained. The production of putrescine and spermidine appears to be initiated by the induction of ornithine decarboxylase which shows a single peak of activity at four hours after hepatectomy. The activity of S-adenosylmethionine decarboxylase shows little change following hepatectomy. The changes in polyamine levels and the activities of the enzymes of polyamine metabolism are not affected by thyroparathyroidectomy 72 hours prior to hepatectomy. Thus although these hypocalcemic conditions considerably reduce and delay DNA synthesis and mitosis, the prereplicative changes in polyamine metabolism still occur. These data suggest that the hepatocytes in hypocalcemic animals have become activated and moved to an advanced stage of prereplicative development before being blocked.     

Coordinated induction and subsequent activity changes of two groups of metabolically interrelated enzymes. Light-induced synthesis of flavonoid glycosides in cell suspension cultures of Petroselinum hortense.

The enzymes of the flavonoid glycoside pathway were specifically induced upon irradiation of a 10-day-old, dark-grown cell suspension culture of Petroselinum hortense Hoffm. with ultraviolet light. The curves for the activity changes of a first sequence of three enzymes (group I) revealed only small, but significant, differences. Sharp peaks in these enzyme activities were observed at about 17, 22, and 23 h after the onset of the irradiation. The apparent half-lives during the subsequent periods of decline ranged, in the same order, from about 10 to 15 and 17 h. No significant differences were found for the lag periods preceding the increases in the three enzyme activities. The possibility is discussed that the slight differences in the patterns of the light-induced activity changes are mainly due to different rates of degradation of the enzymes, suggesting an otherwise largely interpendent regulation. The patterns of the activity changes of four enzymes of the second sequence (group II) differed greatly from those observed for group I, but were again similar to one another. Thus, the two groups of enzymes appear to be regulated differently, despite their concomitant induction. A sigmoidal curve for the accumulation of the flavonoid glycosides was obtained upon the induction of the enzymes. This curve corresponded closely to that derived by integration of the curve for the activity changes of the first enzyme of group I, phenylalanine ammonia-lyase. It is concluded that this enzyme might be rate-limiting for the entire pathway.     

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.     

A distinctive activity of 5'-nucleotidase for dTMP in rat liver mitochondria

The activities of 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5); adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4); AMP deaminase (AMP aminohydrolase, EC 3.5.3.6), and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver 5'-Nucleotidase (5'Nase) and ATP-(Mg2+)-ase activities were compared with similar enzyme activities in the plasma membrane (PM) fraction, obtained from the same biological material. In the regenerating liver, 5'Nase for dTMP diminished its activity by 56% (24 h after partial hepatectomy) and 35 +/- 4% for all substrates in the PM fraction (48 h after operation). In mitochondria, 5'Nase for dTMP manifests sigmoidal substrate activity curve (in contrast with all substrates in the PM fraction and remaining substrates in mitochondria). In vivo 5-azacytidine (a) administered 1 h after partial hepatectomy, prevented changes of 5'Nase activity: (b) administered 24 or 48 h after partial hepatectomy, stabilized low 5'Nase activity (in mitochondria for dTMP, in the PM fraction for all substrates) and decreased ATP-(Mg2+)-ase activity by 51 and 31% in mitochondria and the PM fraction respectively.