Synergistic effects of thyroxine and glucocorticoid in induction of trypsin-like esteroprotease in mouse submandibular gland.

The actions of thyroxine, 5 alpha-dihydrotestosterone and hydrocortisone singly or in combination in enzyme regulation in the submandibular gland were studied in intact and adrenalectomized female mice. 1. Adrenalectomy decreased the activity of trypsin-like esteroprotease (EC 3.4.4.-), and administration of hydrocortisone to adrenalectomized mice restored the activity to normal. 2. Hydrocortisone and thyroxine had synergistic effects in induction of esteroprotease in adrenalectomized mice, but 5 alpha-dihydrotestosterone and hydrocortisone did not have synergistic effects in either intact or adrenalectomized mice. 3. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was not influenced by change in the glucocorticoid level, but was increased by thyroxine and 5 alpha-dihydrotestosterone in both adrenalectomized mice and intact mice.4. Isoelectric focusing in polyacrylamide gel showed that there are three distinct activities of esteroprotease in this gland with isoelectric points of 5.6, 6.2 and 7.3. Both thyroxine and 5 alpha-dihydrotestosterone similarly induced these activities and glucocorticoids did not affected the isozyme patterns induced by the other two hormones.     

Postnatal development of trypsin-like esteroproteases in mouse submandibular gland

The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized naphthyl ester (tosyl-L-lysine alpha-naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.     

Postnatal development of trypsin-like esteroproteases in mouse submandibular gland

Summary The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized napthyl ester (tosyl-l-lysine -naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan     

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.     

Proliferative response of mouse submandibular gland to androgen but not to thyroid hormone

Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and 3H-thymidine labelling index, respectively. Injections of 5 alpha-dihydrotestosterone (4 micrograms/g body weight/day) or L-thyroxine (0.5 microgram/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5 alpha-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of L-thyroxine for any duration did not induce a significant increase in the labelling index. The present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Proliferative Response of Mouse Submandibular Gland to Androgen But Not to Thyroid Hormone

Abstract. Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and ³H-thymidine labelling index, respectively. Injections of 5α-dihydrotestosterone (4 μg/g body weight/day) or l -thyroxine (0.5 μg/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5α-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of l -thyroxine for any duration did not induce a significant increase in the labelling index. the present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Effects of hormonal and nutritional changes on rates of synthesis and degradation of hepatic phosphofructokinase isozymes

The major liver phosphofructokinase isozyme decreased 60–70% in 6-day fasted rats or rats made diabetic with alloxan or streptozotocin. Refeeding induced a return to normal within 72 hr, and insulin treatment for 72 hr increased the amount, of this isozyme 6- to 8-fold greater than the diabetic levels. The level of this isozyme was measured after separation of isozymes on DEAE-cellulose and by titration with antiserum. The minor liver isozyme is slightly, if at all, affected by insulin and only slightly affected during fasting (20% decrease after 6-day fast period).For the fasted rat the results indicated that the decreased amount of the major isozyme was a consequence of increased degradation, with little if any change in the rate of synthesis. However, during refeeding it appeared that the increased enzyme content was a result of both increased synthesis and reduced degradation. Thus. the isozyme representing the bulk of liver phosphofructokinase activity is regulatable through effects on the rates of its synthesis and degradation, while the minor isozyme is relatively unaffected.     

Tissue specificities of protease A-like esteroprotease in male mice

An esteroprotease hydrolyzing p-tosyl-L-arginine methyl ester (TAME) has been purified to homogeneity from male mice submandibular glands by the ammonium sulphate precipitation, Sephadex gel chromatography and DEAE-cellulose chromatography. The enzyme was shown as a single chain acidic protein (pI = 5.7) with the molecular weight of 27.5 K and evidence was obtained to reveal that it was similar to protease A. Using this enzyme as antigen we prepared anti-TAMEase antibody. The immunoblotting studies on tissue specificity using 20 different tissues from male mice revealed that cross-reactivities with anti-TAMEase antibody were observed in the crude extract from the sublingual gland, parotid gland and pancreas. The species specificity studies with the submandibular glands of 7 different species indicated that only the crude extract from rat submandibular glands reacted against anti-TAMEase antibody but it exerted a low TAMEase activity.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland.

Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.     

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.     

Changes in phosphofructokinase isozymes during development of myoblasts to myotubes

The regulation of phosphofructokinase during development of C2C12 myoblasts to myotubes was investigated. Enzyme activity was markedly increased during myogenic development. The increase was observed when enzyme activity was measured under optimal conditions and was not due to changes in the allosteric kinetic properties of the enzyme. Immunoprecipitation of phosphofructokinase from [35S]methionine-labeled myogenic cells revealed that equal amounts of liver and muscle isozymes are present in myoblasts, while in myotubes there was a much higher level of the muscle isozyme. These results were confirmed using an immunoblotting technique. The increase in the level of muscle isozyme in myotubes is due to an increase in the rate of synthesis of the muscle isozyme and occurs in spite of a measurably small increase in its degradation rate. Northern blot analysis using a synthetic oligonucleotide probe showed a 25-fold increase in the level of muscle phosphofructokinase mRNA in myotubes. The conclusion is drawn that the increase in muscle isozyme in myotubes during myogenesis is due to an increase in its mRNA level.     

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.     

Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.     

Kallikrein-like esteroprotease activity and kininase activity in submandibular gland from a mutant mouse with human cystic fibrosis like alterations

The submandibular gland of cri/cri and control mice were compared for their activity of glandular kallikrein like esteroprotease and kininase. Esteroprotease activity is significantly reduced in cri/cri mice with respect to control, with an increased kininase activity in cri/cri mice. Since previous work showed an electrolyte abnormality in the salivary glands of this mutant mouse (1) a possible relationship between this alteration with the low activity of cellular esteroprotease and the high kininase activity is suggested.     

Independent regulation of pyruvate kinase expression by cyclic AMP and prostaglandin F2 alpha in mouse mastocytoma cells

P-815 mouse mastocytoma cells express the K isozyme of pyruvate kinase and the specific activity of this enzyme is increased in response to N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, 8-bromoadenosine 3':5'-cyclic monophosphate, cholera toxin, and epinephrine, all of which also elevate the intracellular concentration of adenosine 3':5'-cyclic monophosphate. Prostaglandin F2 alpha also increases the cellular activity of this enzyme, but does not increase the adenosine 3':5'-cyclic monophosphate levels. Under all these conditions, the increase in enzymatic activity is accompanied by an equivalent increase in the pyruvate kinase protein level. However, neither the rate of enzyme synthesis nor the level of pyruvate kinase mRNA is elevated by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. On the other hand, it does increase the enzyme's half-life. In contrast, prostaglandin F2 alpha increases the rate of synthesis and the level of pyruvate kinase K mRNA, but has no influence on the rate of degradation. Therefore, these cells have two mechanisms which increase pyruvate kinase K levels. One operates via an increase in cAMP level and results in a decrease in the rate of degradation, whereas the other minimizes an upsurge in cAMP levels but still increases pyruvate kinase K activity by increasing its rate of synthesis.     

Parathyroid hormone induction of creatine kinase activity and DNA synthesis is mimicked by phospholipase C, diacylglycerol and phorbol ester

Parathyroid hormone (PTH), which increases cAMP levels, also induces an increase in the activity of the brain isozyme of creatine kinase and in DNA synthesis in osteoblast-enriched bone cell cultures by a cAMP-independent mechanism. The following results lead us to the conclusion that PTH induction of brain isozyme of creatine kinase activity and DNA synthesis occurs by activation of membranal phospholipid metabolism leading to increased protein kinase C activity and Ca2+ mobilization, a mechanism demonstrated for several growth factors and other hormones. (1) Binding of membranal phospholipids by agents such as gentamycin or antiphospholipid antibodies abolishes the stimulation by PTH of creatine kinase activity and DNA synthesis but not of cAMP production. (2) Treatment of cell cultures with exogenous phospholipase C increases brain isozyme of creatine kinase activity and DNA synthesis, but not cAMP production; these stimulations are also blocked by serum containing anti-phospholipid antibodies. PTH has no additional effect on stimulation of creatine kinase activity by phospholipase C (and only a slight effect on DNA synthesis). (3) A synthetic diacylglycerol (1-oleyl-2-acetyl glycerol) or phorbol ester (phorbol 12-myristate 13-acetate) or Ca2+ ionophore, A23187 induces creatine kinase activity and DNA synthesis in the cultures. However, this effect is not blocked by antiphospholipid sera and PTH has no additional effect. (4) Inhibition of protein kinase C activity by drugs reported to inhibit the enzyme (retinoic acid, quercetin) abolishes the stimulation of brain isozyme of creatine kinase activity and of DNA synthesis by PTH.     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.     

Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagine-linked glycoproteins in rat mammary gland

Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.