Mammary Gland Microsomes Research Articles

Human Growth Hormone Deletion Mutant (hGH44-191) Binds with High Affinity to Lactogenic Receptors but Not to Somatogenic Receptors

The carboxyl-terminal hGH fragment, hGH44–191, displays diabetogenic activity. To understand whether this biological activity is mediated through somatogenic or lactogenic receptors, we investigated the ability of hGH44–191to bind both receptor classes. We found that hGH44–191could not compete with [125I]hGH or [125I]bGH for bovine liver somatogenic binding sites. Additionally, hGH44–191could not displace [125I]hGH from the somatogenic receptor sites when human liver microsomes were used as the receptor source. In contrast, hGH44–191effectively competed with [125I]hGH for lactogenic receptor sites of lactating mammary gland microsomes and of bovine liver microsomes. In summary, hGH44–191does not bind to somatogenic receptors but does not bind to lactogenic receptors. These data suggest that the biological actions of hGH44–191could be mediated through lactogenic receptors.

Debenzoylating and deacetylating activities of rat liver and mammary gland microsomes: Effect of ovariectomy

This study compared the rates of N-deacylations of N-hydroxy- N-2-fluorenylbenzamide (N-OH-2-FBA) with those of its analogue, N-hydroxy- N-2-fluorenylacetamide (N-OH-2-FAA), by the mammary gland (tumor target for both compounds) and the liver of female Sprague-Dawley rats and examined the effect of ovariectomy on these activities. N-Debenzoylation of N-OH-2-FBA was catalyzed by the mammary and liver microsomes of 50-day-old female rats at similar rates (~24 nmol/min/mg). The activity of both tissues increased (up to 1.8 times) after ovariectomy at 42, 32 and, especially, 22 days of age. The rapid hydrolysis appeared to be unique for the benzoyl group since N-OH-2-FAA was deacylated only ~0.05 and 0.004 times as fast by the liver and mammary microsomes, respectively, and these low rates were unaffected by ovariectomy. Since such substrate specificity would be of significance in the metabolism of xenobiotics and drug design, esterase activity and its sensitivity to ovariectomy at 22 days of age were examined with several acetylated and benzoylated substrates in the liver and mammary microsomes and compared with those of male liver. Tissues of rats of both sexes had a greater capacity to hydrolyze carboxyl esters than amides. Except for N-2-fluorenylacetamide (2-FAA) and o-nitrophenylacetate (o-NPA), all substrates were hydrolyzed by liver microsomes of the male up to 3.9 times faster than by those of the female. Microsomes of female liver hydrolyzed acetylated substrates 1.2 to 25 times faster than benzoylated analogues except for N-OH-2-FBA and benzamide. By contrast, mammary gland microsomes hydrolyzed benzoylated compounds 1.4 to 333 times faster except for 2-naphthyl benzoate. Respective rates of hydrolysis of o-NPA by microsomes of liver and mammary gland were 1.7 and 0.6 times those of p-NPA. After ovariectomy, deacylating activities increased (up to 1.6 times) except for those of 2-FAA and acetanilide. All deacylations were > 98% inhibited by 0.1 mM paraoxon, indicating catalysis by serine hydrolases. The results suggest involvement of multiple carboxylesterases and indicate that certain benzoylated xenobiotics may have a greater effect on the mammary gland than acetylated xenobiotics because of their greater vulnerability to hydrolysis by esterases of mammary gland.

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in fried beef, is mammary gland carcinogen in rats. Using the 32P-postlabeling method, PhIP-DNA adduct levels were measured in mammary epithelial cells isolated from female Sprague-Dawley rats given 10 daily doses of PhIP (75 mg/kg, p.o.) according to a protocol previously shown to induce mammary gland cancer. At 24 h, 48 h, 1 week and 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SD] were 10.2 +/- 0.7, 7.9 +/- 2.7, 2.2 +/- 0.6 and 0.9 +/- 0.03 respectively. When isolated rat mammary epithelial cells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP (45 microM, 1 h, 37 degrees C), PhIP-DNA adducts were detected in cell DNA (RAL = approximately 97 x 10(7); however, no adducts were detected in cells incubated with PhIP (200 microM, 15 h, 37 degrees C). Incubating cells with pentachlorophenol, an inhibitor of acetyltransferase, or incubating cells at 0-4 degrees C, reduced N-hydroxy-PhIP adduct levels by 45 and 75% respectively, indicating that formation of N-hydroxy-PhIP adducts was largely due to metabolic activation. Further studies showed that rat mammary gland microsomes had little capacity to N-hydroxylate PhIP, as assayed by the mutagenic activation of PhIP in the Ames Salmonella assay. In contrast, N-hydroxy-PhIP was metabolically activated by cytosol-catalyzed PhIP-DNA adduct formation to calf thymus DNA incubated in vitro with N-hydroxy-PhIP (2 microM) in the presence of acetyl CoA. Notably, mammary cytosolic O-acetyltransferase activation of N-hydroxy-IQ or N-hydroxy-MeIQx. All three N-hydroxylamines were activated via cytosolic proline aminoacyl-tRNA synthetase and phosphorylase, although the activities of these enzymes were approximately 100-fold lower than O-acetyltransferase. No mammary cytosolic sulfotransferase activation could be detected with any of the N-hydroxylamines. Our results are consistent with the notion that PhIP-DNA adduct formation and initiation of carcinogenesis in the rat mammary gland may be associated with N-hydroxylation of PhIP outside the mammary gland, transport of the N-hydroxylamine to the mammary gland and subsequent in situ O-acetyltransferase-catalyzed activation of N-hydroxy-PhIP.

Metabolic activation of heterocyclic amine food mutagens in the mammary gland of lactating Fischer 344 rats

We investigated the ability of the mammary gland to metabolically activate 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5- f]-quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Although mammary gland microsomes had almost no capacity to metabolically activate the parent compounds, mammary gland cytosol was able to esterify the N-hydroxylamines. Acetyltransferase was the primary enzyme responsible for the phase II activation of the N-hydroxylamines. The level of acetyl CoA-stimulated binding when N-hydroxy PhIP served as the substrate was approximately 3- and 17-fold higher than when IQ and MeIQx served as substrates, respectively. N-Hydroxy-IQ and N-hydroxy PhIP can also be activated by tRNA synthetase and phosphatase, but not by sulfotransferase. However, the levels of proline- and ATP-enhanced DNA binding was approximately 30- and 60-fold lower than the acetyl CoA-enhanced DNA binding of IQ and PhIP, respectively. Differences observed in the phase II activation of the various heterocyclic amines in the mammary gland may explain why the mammary gland is a target organ for PhIP-induced carcinogenicity but not for IQ- or MeIQx-induced carcinogenicity in Fischer 344 rats.

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes

Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same K m (2 μ m retinol) between tissues but a higher V max in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterifled in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar V max (300 pmol/min/mg microsome protein) between tissues but K m values of 15 and 5 μ m for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 μ m, respectively, in the liver. Thus, when substrate was near or below K m , retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 μ m in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esteriflcation is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.

Characterization of insulin binding to bovine liver and mammary microsomes

1. 1. Bovine liver and mammary gland (MG) appear metabolically independent of insulin, yet the specificity and kinetics of 125I-insulin ( 125I-INS) binding to bovine liver and MG microsomes (MIC) indicate the presence of insulin receptors in MIC from both tissues. 2. 2. The insulin receptors from bovine liver ( K d = 7.6 × 10 −10 M) and MG ( K d = 9.6 × 10 −11 M) were similar to each other and to other insulin receptors in their binding affinities and pH optima. 3. 3. Perturbation of rat liver and bovine MG MIC by phospholipase or NaCl treatment increased 125I-INS binding to the membranes, suggesting exposure of cryptic insulin receptors. 4. 4. Different responses in 125I-INS binding to membrane perturbation suggest differences between rat and bovine membranes.

Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling.

The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with [125I]ovine PRL [( 125I]oPRL). PRL receptors were solubilized from mammary microsomes with 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of [125I]oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five [125I]oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit. From these observations, we conclude that this predominant 32,000 mol wt component is a major binding subunit of the PRL receptor molecule and does not aggregate with itself or with other subunits through S-S linkages.

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. II. Glucuronidation of ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide

We determined UDP-glucuronyltransferase (UDP-GT) activities of hepatic and mammary gland microsomes of female rats with p-nitrophenol and the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide (2-FAA) and the effects of hepatic inducers of UDP-GT's on these glucuronidations. Pre-treatment of non-lactating (NL) and lactating (L) rats with beta-naphthoflavone (beta-NF) significantly increased glucuronidations, of p-nitrophenol, the phenolic metabolites of 2-FAA, especially of 5-hydroxy-2-FAA, and also of N-hydroxy-2-FAA by hepatic microsomes. Pre-treatment of L rats with beta-NF or 3-methyl-cholanthrene (3-MC) significantly increased glucuronidations of these compounds by mammary gland microsomes suggesting that both liver and mammary gland of L rats possess similar UDP-GT activities. In NL rats, UDP-GT activities of mammary microsomes toward phenols were greater than in L rats, and except for that of 5- and 7-hydroxy-2-FAA, were not inducible with beta-NF. The data obtained with L rats, the greater magnitude of stimulation of the hepatic UDP-GT of NL rats by beta-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT of mammary microsomes suggested that the phenolic metabolites of 2-FAA and N-hydroxy-2-FAA share chiefly the characteristics of substrates for group 1 UDP-GT activities (i.e., those inducible with beta-NF or 3-MC). Neither inducer increased glucuronidation of 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT of mammary or hepatic microsomes. In contrast to hepatic microsomes which formed considerable amounts of the glucuronide of N-hydroxy-2-FAA, mammary gland microsomes glucuronidated this substrate to a minor extent only. This suggested that glucuronide of N-hydroxy-2-FAA may play a role in systemic, but not in local mammary tumorigenesis by N-hydroxy-2-FAA.

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide.

We determined ring- and N-hydroxylations of a systemic mammary gland carcinogen, N-2-fluorenylacetamide (2-FAA), by microsomal fractions of liver and mammary gland of female rats and the effects of in vivo and/or in vitro modifiers of these oxidations. Pretreatment of lactating rats with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF) and non-lactating (50-day old virgin) rats with beta-NF showed similar effects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAA by hepatic microsomes was increased manyfold and the formation of 1-hydroxy-2-FAA was induced. In mammary gland microsomes, the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased, but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomes of lactating rats had capacity for N-hydroxylation which was increased approximately 3 times by pretreatment of rats with 3-MC or beta-NF. All of the induced increases of metabolites of 2-FAA in hepatic and mammary microsomes were inhibited by 0.1 mM alpha-naphthoflavone (alpha-NF) in vitro. Pretreatment of non-lactating rats with phenobarbital increased only the formation of 7-hydroxy-2-FAA in hepatic microsomes which was further stimulated by alpha-NF in vitro. The latter also stimulated the formation of 7- and 9- hydroxy-2-FAA by hepatic microsomes of the uninduced rats, but had no effects in mammary microsomes, in which 9-hydroxy-2-FAA was a major metabolite. Hence, the data showed qualitative and quantitative differences between lactating and non-lactating rats in metabolism of 2-FAA by mammary microsomes which may result from differences in the levels (e.g., of cytochrome P-450) and activities of microsomal enzymes determined herein. In hepatic microsomes of these rats, differences in quantities of metabolites of 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in corn oil-treated rats only. The solvent (methanol or acetone) used for addition of 2-FAA to the incubation mixtures altered quantitatively the metabolite profiles in hepatic and mammary microsomes of 3-MC or beta-NF treated rats. The formations of 1- and 3- or 5- and 7-hydroxy-2-FAA were greater in the presence of acetone or methanol, respectively. The results of this study suggest that the formation of phenolic and N-hydroxy metabolites of 2-FAA in both hepatic and mammary microsomes of lactating rats is catalyzed by similar form(s) of cytochrome P-450 induced by pretreatment with 3-MC or beta-NF.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholesterol esterification by mammary gland microsomes from the lactating rat.

Cholesterol esterification by fatty acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) has been demonstrated in microsomes prepared from breast tissue of the lactating rat, employing the incorporation of [1-14C]oleoyl coenzyme-A into cholesteryl[14C]oleate. The regulation of this activity in vitro was studied by supplementing microsomes with unesterified cholesterol supplied either as a dispersion in acetone or by incubating microsomes with cholesterol-enriched serum lipoproteins prior to enzyme assay. ACAT activity was increased by more than 80% when the ratio of unesterified cholesterol to microsomal protein was increased from 29 to 48 micrograms/mg protein, indicating that the normal cholesterol content of mammary gland microsomes does not saturate this enzyme. Cholesterol esterification could be inhibited nearly completely in vitro by addition of the polar steroid progesterone (93% decrease in ACAT activity with 75 microM progesterone) and, to lesser extents, by estradiol and retinol. Enzyme assays were also performed after incubating microsomes with N-acylamides that inhibit cholesteryl ester synthesis in other systems. Under conditions where the mammary gland ACAT reaction was inhibited by more than 82%, the esterification of retinol was reduced by less than 30% and incorporation of [14C]oleoyl-CoA into triglycerides was not inhibited at all. These studies indicate that the lactating mammary gland contains ACAT activity having properties similar to ACAT in other organs. The presence of ACAT activity in the lactating mammary gland provides a possible mechanism for the synthesis of cholesteryl esters found in milk. It can also be inferred that ACAT in mammary gland microsomes is likely to be distinct from the microsomal acyltransferases that catalyze the esterification of retinol and glycerides.

Glycosyl transferases in mouse and human milk fat globule membranes.

Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats: Effects of 3-methylcholanthrene and β-naphthofla vone

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b 5, NADH- and NADPH-dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa 3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa 3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-448) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and β-naphthoflavone, increased the cytochrome content 3- to 10-fold and 2-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[ a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 mM α-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.

Retinol esterification by mammary gland microsomes from the lactating rat.

Because vitamin A in milk is largely present as esterified retinol while blood plasma predominantly contains unesterified retinol, experiments were conducted to determine whether membranes from the lactating mammary gland are able to synthesize retinyl esters in vitro. When microsomes from rats lactating for 7 to 14 days were incubated with [(3)H]retinol dispersed in dimethylsulfoxide, some [(3)H]retinol esterification was observed (147 pmol/5 min per 0.5 mg protein). However, 3- to 7-fold increases in retinyl ester synthesis could be achieved by supplying either a fatty acyl CoA-generating system or preformed fatty acyl CoA thioesters; thus, the major activity in vitro has the characteristics of a fatty acyl CoA: retinol acyltransferase. Both long-chain and medium-chain fatty acyl CoA esters stimulated [(3)H]-labeled retinyl ester synthesis in vitro. Concordantly, analysis of the retinyl ester pattern of rat milk demonstrated the presence of eight different esters of retinol ranging in fatty acyl chain length from 8 to 18 carbons. Retinol esterification by microsomes was maximal at neutral pH (7.1) in the presence of approximately 50 micro M palmitoyl CoA, and was linear with time of incubation for at least 5 min. Retinyl ester synthesis increased with the apparent concentration of [(3)H]retinol to approximately 200 nmol/ml, but was also dependent on the ratio of retinol relative to total microsomal protein in the incubation mixture. These experiments demonstrate for the first time retinol esterification by mammary gland membranes and point to the hypothesis that free retinol from plasma is esterified in this organ before secretion of retinyl esters in milk.-Ross, A. C., Retinol esterification by mammary gland microsomes from the lactating rat.

Rat mammary gland carcinogenesis after local injection of N-hydroxy-N-acyl-2-aminofluorenes: relationship to metabolic activation

A single local injection of 2.5 mumol of N-hydroxy-N-formyl-2-aminofluorene (N-hydroxy-FAF), N-hydroxy-N-acetyl-2-aminofluorene (N-hydroxy-AAF), or N-hydroxy-N-pro-pionyl-2-aminofluorene )N-hydroxy-PAF) to each of the six left mammary glands of female Sprague-Dawley derived CD rats gave a mammary tumor incidence, after 12 months, of 53% for the N-acetyl (42% adenocarcinoma, 11% fibroadenoma), 41% for the N-formyl (8% adenocarcinoma, 11% sarcoma, 22% fibroadenoma), and 33% for the N-propionyl (11% adenocarcinoma, 22% fibroadenoma) derivatives of N-hydroxy-N-2-aminofluorene, Latent periods for malignant tumor appearance (adenocarcinoma or sarcoma) was 210 days, 148 days, and 177 days, respectively, with no malignant tumors occurring in the vehicle-treated animals. In contrast, latent periods for benign tumor appearance (fibroadenoma) was 263 days for control animals, 289 days for the N-hydroxy-AAF, 324 days for the N-hydroxy-FAF, and 317 days for the N-hydroxy-PAF animals. When N-acetyl-2-aminofluorene (AAF) was applied as above there was only an 8% mammary tumor incidence (4% adenocarcinoma, 4% fibroadenoma) with a latent period of 207 days for malignant tumor (adenocarcinoma) and 221 days for benign tumor (fibroadenoma) appearance. Arylhydroxamic acid N, O-acyltransferase activity has been demonstrated in the mammary glands of male, and lactating and non-lactating female Sprague-Dawley derived CD rats by means of nucleic acid binding assay. Mammary gland cytosol catalyzed tRNA adduct formation to a greater extent with N-hydroxy-FAF. AAF was not activated by this enzyme. Ammonium sulfate fractionation demonstrated the presence of two enzymes, one specific for N-hydroxy-FAF (70-80% fraction), the other specific for N-hydroxy-AAF and N-hydroxy-PAF (40-70% fraction). Moreover, gel filtration chromatography of mammary gland cytosol demonstrated the presence of two enzymes of differing acyl specificity. Mammary gland microsomes catalyzed the formation of tRNA adducts, but only with the N-hydroxy-FAF derivative. Assays that tested the mutagenic potential of the arylhydroxamic acids in Salmonella typhimurium TA-1538 with either mammary gland cytosol or microsomes demonstrated the order of mutagenicity to be N-hydroxy-FAF greater than N-hydroxy-AAF greater than N-hydroxy-PAF. A similar order of mutagenicity was demonstrated without an external metabolic activation system. These data demonstrate that the presence of two distinct enzymes in the rat mammary gland that activate arylhydroxamic acids.

Triglyceride Biosynthesis by the sn-Glycerol-3-Phosphate Pathway in Milk Cytoplasmic Droplets

Enzyme preparations similar in their properties to those of mammary gland microsomes have been obtained by ultra-sonication of cytoplasmic droplets from freshly-secreted milk from goats. Enzymes of the sn-glycerol-3-phosphate pathway in milk have been confirmed through such preparations in vitro.

Kinetics of a micelle specific palmitoyltransferase isoenzyme of rabbit mammary gland.

Palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmitoyltransferase [EC 2.3.1. -] in rabbit mammary gland microsomes is composed of two isoenzymic species. The alpha form (LPAT-alpha) is active with monomeric substrates and inhibited by micelles while the beta form (LPAT-beta) is active only with micelles. By combining the effects of time, temperature, and Tween 80 which selectively inhibited LPAT-alpha, the substrate saturation curve for the LPAT-beta isoenzyme has been successfully determined. Both theoretical and experimental curves are in good agreement.

Isoenzymes of an acyltransferase from rabbit mammary gland: Solubilization of the micelle-specific species with Triton X-100

The micelle-specific palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmityltransferase isoenzyme from lactating rabbit mammary gland microsomes is selectively solubilized in Triton X-100 but not Tween 80. Both detergents inactivate the monomer-specific isoenzyme. The possibility that, in vivo , physiological surfactants regulate the relative activities of these two isoenzymes is discussed.

Fatty acid desaturation by mammary gland microsomes from lactating mice.

AbstractMicrosomal fractions obtained from lactating mouse mammary glands catalyze the desaturation of myristic, palmitic, and stearic acids in the presence of adenosine 5′‐triphosphate, coenzyme A, and nicotinamide adenine dinucleotide, reduced form. The products of this reaction are the corresponding monoenoic fatty acids, myristoleic, palmitoleic, and oleic acids respectively. Desaturase, activity was greater with stearate as substrate than with either palmitate or myristate. Palmitate was desaturated at a rate ca. 50% greater than that of myristate. Although both nicotinamide adenine dinucleotide, reduced form and nicotinamide adenine dinucleotide phosphate, reduced form served as electron donors, the former was superior in this regard. Desaturase activity was not influenced by the addition of glycerol 3‐phosphate to the incubation medium. However, the presence of this compound did affect the type of lipid formed. In the absence of the added glyceride‐glycerol precursor, the major products were phospholipids, whereas in its presence they were triglycerides.

Ribonuclease in bovine milk

The ribonuclease of milk has not been investigated, although there has been in recent years a rapid expansion of our knowledge of ribonuclease in body fluids such as cerebral spinal fluid ( Houck, 1958), blood ( Levy and Rottino, 1960; Rabinovitch and Dohi, 1957; Zittle and Reading, 1945), urine ( Levy and Rottino, 1960), and the extracellular fluid of skin ( Tabachnick and Freed, 1961). Zittle and DellaMonica (1952) noted that certain purified fractions of bovine milk showed phosphodiesterase activity when ribonucleic acid was used as the substrate. Bailie and Morton (1958) showed that the nucleic acid content of mammary gland microsomes diminished when the microsomes were incubated in milk serum for 12 hours, and suggested that the phosphodiesterase of Zittle and DellaMonica (1952) might have caused the decrease in nucleic acid. They suggested that milk microsomes (with a low nucleic acid content) might be derived from mammary gland microsomes, which contained a much higher nucleic acid content. In this report evidence is presented for the presence of relatively high concentrations of ribonuclease in cow's milk. Some properties of this enzyme, as well as its partial purification, are described.