Presence of cytosolic phosphoenolpyruvate carboxykinase activity in rat mammary gland

The cytosolic P-enolpyruvate carboxykinase activity was measured in mammary gland and liver of animals at all stages of the reproductive cycle. The specific activity of this enzyme was almost absent in the mammary gland of virgin rats. Different pattern of P-enolpyruvate carboxykinase activity was obtained in liver and mammary gland during lactation cycle. The specific activity of the enzyme increased more than 40-fold in mammary gland and 2-fold in liver during the transition from mid-pregnancy to mid-lactation. Weaning of mid-lactating rats for 48 h resulted in an abrupt change in the enzyme activity in mammary gland while there was a small change in liver. In all the experiments performed, the activity of P-enolpyruvate correlates inversely with the plasma insulin levels described for the lactogenic process.     

Propionyl-CoA synthetase in mammary gland and liver of cows

Propionyl-CoA synthetase of liver and mammary gland from calf and midlactation cow was investigated. No activity of this enzyme was detected in calf mammary gland, but it was detected in calf liver. Propionyl-CoA synthetase was found in both, liver and mammary gland of the cow, although mammary gland activity was about 25% of that found in liver. The effects of pH and temperature on enzyme activity and stability were also investigated in crude extracts of liver and mammary gland tissues. The results suggest a different behaviour of the enzyme from both origins. Kinetic studies of the enzyme were also carried out, showing differences, depending on the organ, in the apparent substrate KM values.     

Regulation of cholesterol synthesis in the liver and mammary gland of the lactating rat.

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) in the lactating mammary gland of rats killed between 10:00 and 14:30 h was 2-3 times that in the livers of the same animals. In contrast, after injection of 3H2O in vivo, the rate of appearance of 3H in the cholesterol of the gland was much lower than that in the liver. In the mammary gland of virgin and non-lactating animals, the activity of HMG-CoA reductase was less than 10% of that of the lactating gland. The activity of HMG-CoA reductase in the lactating mammary gland was significantly (P less than 0.005) lower at midnight than at mid-day, and appeared to show an inverse relationship to the activity of the liver enzyme. However, there was no corresponding change in the incorporation of 3H into the gland cholesterol. Withdrawal of food for 6h had no effect on the activity of HMG-CoA reductase in the lactating mammary gland, but resulted in a significant decrease (P less than 0.005) in that of the liver. Starvation of lactating rats for 24h produced a significant decrease (P less than 0.005) in the activity of the enzyme in both organs. There was also a significant decline in the rate at which 3H2O was incorporated in vivo into the cholesterol of both organs (liver, P less than 0.05; gland, P less than 0.005). Giving a high-fat palatable diet together with chow to lactating animals led to a decline in HMG-CoA reductase activity in the mammary gland, but not in liver. This decrease in the gland was not accompanied by a corresponding decline in the apparent rate of cholesterol synthesis.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Polyamine biogenesis in the rat mammary gland during pregnancy and lactation

Polyamines and RNA accumulate in the rat mammary gland during pregnancy, but the major increases occur after parturition. Therefore the major increases occur after the gland has obtained its maximal complement of epithelial cells. During lactation, the spermidine concentration rises above 5mm and RNA content in the lactating mammary gland reaches a value 16 times that of the unstimulated mammary gland. The ratio of spermidine/spermine, an increase of which initially signals an elevation in biosynthetic activity, is near 1 in the normal mammary gland and is greater than 10 in the lactating mammary gland. Putrescine concentration is very low during the entire course of mammary-gland development, with the exception of early pregnancy. The low putrescine concentration probably reflects the very rapid conversion of putrescine into spermidine. Both ornithine decarboxylase, the enzyme that synthesizes putrescine, and putrescine-stimulated S-adenosyl-l-methionine decarboxylase, the enzyme that synthesizes spermidine, increase in activity during middle and late pregnancy; during lactation, both enzyme activities are elevated until the 21st day of lactation, and then decline. These declines are concomitant with involution. Also, it was found that the amount of ribonuclease activity in the mammary gland was very high during lactation, almost double that in the gland during pregnancy.     

Comparison of alkaline phosphatase activity in normal, lactating and neoplastic rat mammary tissue

1. Alkaline phosphatase activity in NMU-induced rat mammary tumours was compared with activity in normal and lactating mammary gland. 2. Both tumour and normal mammary alkaline phosphatase were sensitive to heat inactivation and inhibition by phenylalanine. 3. Specific activity of enzyme in tumours was comparable to normal mammary tissue. 4. Mammary gland alkaline phosphatase increased markedly in late pregnancy and early lactation. 5. Bromocryptine treatment had no effect on enzyme activity in lactating mammary gland.     

Medium chain acyl-thioester hydrolase activity in goat and rabbit mammary gland fatty acid synthetase complexes

The goat mammary gland fatty acid synthetase hydrolysed both medium (C_8:0, C_10:0) and long (C_16:0, C_18:0) chain length acyl CoA esters, whereas the enzyme from rabbit mammary gland only hydrolysed long chain length acyl CoA esters. The medium chain acyl-thioester hydrolase activity of goat mammary gland fatty acid synthetase was much less sensitive to inhibition by phenylmethanesulfonyl-fluorid than the long chain acylthioester hydrolase activity. These results indicate the presence of either two acyl-thioester hydrolases with different specificity or one acyl-thioester hydrolase containing two different active sites.     

Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.     

Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

The difference between cytosol and membrane growth-related protein kinase activities in lactating mouse mammary gland

1. A protein kinase activity which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary gland cells was isolated and partially purified from cytosol. 2. Another protein kinase activity was demonstrated in crude membranes of lactating mouse mammary gland. 3. By the use of several different synthetic peptides as a substrate, it was demonstrated that the cytosol enzyme was a serine kinase, while the membrane protein kinase activity was mainly due to tyrosine kinase.     

Specificity of diacylglycerol acyltransferase from bovine mammary gland, liver and adipose tissue towards acyl-CoA esters.

1. Microsomal diacylglycerol acyltransferase from bovine lactating mammary gland, liver and adipose tissue was capable of acylating microsomal-bound 1,2-dipalmitolyglycerol with acyl-CoA of chain length C4--C18. 2. The activity of the liver and adipose enzymes towards butyryl-CoA and hexanoyl-CoA relative to longer-chain acyl-CoA was similar to that of the mammary enzyme. The Km and V values of the three enzymes with butyryl-CoA and hexanoyl-CoA were similar, except for the V values of the adipose enzyme which were higher. 3. Microsomal diacylglycerol acyltransferase from mammary gland and liver of non-ruminants was also capable of utilizing butyryl-CoA. 4. These results indicate that the unique presence of short-chain acids in ruminant milk triacylglycerols is not caused by differences in specificity between the diacylglycerol acyltransferase from ruminant mammary and other tissues.     

The activity of phosphoenolpyruvate carboxykinase throughout the lactation cycle of the guinea pig mammary gland

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) has been measured in the guinea pig mammary gland throughout the pregnancy-lactation cycle. This is of interest since the primary importance of PEPCK is thought to be its role in gluconeogenesis and it is questionable whether or not gluconeogenesis occurs in the mammary gland. The enzyme activity, present in both the cytosol and mitochondria, was shown to follow the lactation profile. During the transition into lactation, cytosolic PEPCK activity increases 11-fold and mitochondrial PEPCK activity 43-fold while tissue weight increases 4-fold. Fructose 1,6-bisphosphatase was found to increase at a rate only slightly greater than that of the tissue weight. The increase in mitochondrial PEPCK activity is thus about 10 times greater than that of general tissue expansion, whereas the cytosolic PEPCK activity increase is only 2-fold greater. The activity of fructose 1,6-bisphosphatase appears to be merely keeping pace with general tissue expansion. The mitochondrial enzyme constitutes 59 +/- 3% of the total gland PEPCK activity in the prepartum state and 86 +/- 2% at midlactation. Therefore, mitochondrial PEPCK is the isoenzyme undergoing the greater increase during the transition into lactation in the guinea pig mammary gland and thus would appear to play the more important role in the conversion of oxalacetate to phosphoenolpyruvate in this tissue.     

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Intranuclear dynamics of DNA polymerase α differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase α activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase α activity, with the majority (60–75%) localized in soluble nucleoplasm and a significant amount (13–20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase α was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60–80%) of DNA polymerase α activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60–80%) of DNA polymerase β activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase α molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase α which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase β in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Changes in fat content and some characteristics of lipolytic activity during pregnancy and lactation in mouse mammary gland.

The amount of free fatty acid in the mouse mammary gland continuously increased throughout pregnancy and lactation, while the amount of triglyceride which had been stored in the gland rapidly decreased after parturition. Higher lipolytic activity in the gland was observed in pregnancy than in nonpregnant and lactating animals. The optimum pH of the activities before and after parturition were about 6 and 7, respectively, and the activities did not decrease at high ionic strength in contrast to the ion dependent inactivation described in lipoprotein lipase. Incubation of the enzyme extract of the lactating mouse mammary gland at 50 degrees C for 10 min led to a remarkable increase in the lipolytic activity measured at pH 6.0, suggesting the existence of either an inactive form of the lipase whose optimum pH is 6.0 or some heat sensitive inhibitor(s) or inactivator(s) of the enzyme in the lactating mammary gland. The triglyceride stored in the gland in pregnancy will be consumed within the first 3rd days after parturition, and the lipases play an important role in the decomposition of the triglyceride.     

Hormonal activation of mammary gland peroxidase.

Ultrastructural cytochemistry was used to detect an endogenous peroxidase in the rat mammary gland. The enzyme was identified only during the latter half of pregnancy and during lactation, indicating its possible dependence upon hormones. To test this hypothesis, specific hormones associated with the development and differentiation of the mammary gland were used both in vivo and in vitro in an effort to induce, or unmask, the activity of the enzyme. Estrogen injected into nonpregnant rats induced some peroxidase activity in the mammary glands of a few animals. Two hormone combinations tested in organ cultures of mouse mammary gland were able to activate the enzyme: (1) dexamethasone + insulin and (2) dexamethasone + insulin + prolactin.     

Effects of variations in thyroid hormone serum concentrations on serum ACE-activity

Serum angiotensin converting enzyme activities were significantly increased in 26 untreated hyperthyroid patients (20.3 +/- 5.4 U/ml; P less than 0.001) compared with healthy control subjects (13.1 +/- 2.3 U/ml). In 12 patients a significant fall in enzyme activities was observed after treatment compared with pretreatment serum ACE levels (P less than 0.001). Eight patients with hypothyroidism (15.7 +/- 5.1 U/ml) and 11 athyreotic patients, totally thyroidectomized for well-differentiated thyroid cancer, showed no significant differences in serum ACE activities (14.3 +/- 2.2 U/ml) compared with control subjects. After thyroid hormone supplementation a significant increase in serum ACE activity (P less than 0.05) was found in the athyreotic patients. Addition of increasing amounts of L-thyroxine to a serum sample of an athyreotic patient showed no significant effect on ACE activity in vitro. We suggest that the elevated serum ACE activity in hyperthyroidism is not from the thyroid gland, but represents a direct effect of thyroid hormone on ACE synthesis and/or release from endothelial cells.     

Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland

A serine-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated Mr 74 000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X-100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.     

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states.

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland). It was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.