Raoul Carubelli

Neuraminidase activity in brain and liver of rats during development.

Abstract 1. 1. A soluble neuraminidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) with pH optimum 5.8 and particle-bound neuraminidase with pH optimum 4.4. are present in brain and liver of developing rats. 2. 2.The properties (i.e. pH optima, effect of mono and bivalent cations and substrate specificity) of these two enzymes in both organs are essentially identical to those previously reported for the enzymes present in the cytosol and in the lysosomes, respectively, of mature rat liver. 3. 3. The developmental patterns of the soluble neuraminidases of brain and liver are quite different. Brain has very low levels of enzyme activity during the first week of life, followed by a gradual increase which starts on the second week and levels off toward the end of the third week of postnatal development. The liver enzymes, which is fully activate at birth but very low in fetal liver, exhibits a sharp increase during late prenatal development, 3–4 days prior to birth. 4. 4. The specific activities of the particle-bound neuraminidases of both organs remain at a fairly constant level during the period from 1 week before birth to 1 month of age.

Isolation and characterization of glycoproteins from canine tracheal mucus

Three homogeneous glycoproteins were isolated from reduced and S-carboxy-methylated canine tracheal pouch mucus by gel filtration and ion-exchange chromatography. Initial fractionation was carried out on Sephadex G-200; chromatography of the excluded Sephadex G-200 fraction on Bio-Gel A-15 m yielded two high molecular weight glycoprotein fractions. Following rechromatography on the same column, the main fraction behaved as an electrophoretically homogeneous high molecular weight (581 600) glycoprotein, with a high carbohydrate content (80%) and a single amino-terminal amino acid (arginine). Ion-exchange chromatography (DEAE-cellulose) of the included Sephadex G-200 fraction yielded two electrophoretically homogeneous glycoproteins of lower molecular weight (20 800 and 24 600, respectively). A single amino-terminal amino acid, glycine and alanine, respectively, was detected for each glycoprotein. Chemical analysis of these three glycoproteins revealed the presence of fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid and sulfate monoester. The high molecular weight glycoprotein had a higher hexose, sialic acid and sulfate content, per mg of protein, than the low molecular weight glycoproteins. The results of the alkaline borohydride treatment indicated that the majority of the carbohydrate chains of these glycoproteins are linked to the protein core through O-glycosidic bonds involving N-acetylgalactosamine and serine or threonine.

Effect of Oxygen Free Radicals on Corneal Collagen

Corneal collagen was labeled in vivo by injection of 14C-proline into the anterior chamber of rabbit eyes. The isolated corneal collagen was incubated in iron-free phosphate buffered saline (pH 7.4) containing 1 mM ascorbate and 0.1 mM CuSO4 for either 1 hour or 3 hours at 37 degrees. Addition of 2 volumes of 8M urea-1 mM dithiothreitol and heating for 1 min at 100 degrees solubilized virutually all of the collagen in the control incubations but left a significant amount of insoluble collagen in specimens exposed to the hydroxyl radical generating system. This residue amounted to 19% and 38% of the initial radioactivity in samples incubated for 1 h and 3 h, respectively. The chromatographic profiles (gel filtration on CL-4B) of the soluble fraction showed an increase in both aggregation and degradation products of collagen in the 1 h incubation mixture, whereas after 3 h there was an increase only in degradation products. These observations suggest that additional crosslinking of the soluble collagen aggregates observed at 1 h may be responsible for their subsequent disappearance at 3 h, with concomitant increase of the insoluble fraction. Collagen degradation by .OH may play a role in corneal ulceration, whereas hydroxyl radical-mediated crosslinking is consistent with age-dependent increases in insoluble collagen.

Studies on the soluble and lysosomal neuraminidases of rat mammary glands

Abstract 1. 1. Rat mammary glands contain a soluble neuraminidase ( N- acetylneuraminate glycohydrolase, EC 3.2.1.18) in the cytosol and a particulate neuraminidase strongly bound to the lysosomes. The pH optima were 5.8 for the soluble and 4.4 for the lysosomal enzyme. 2. 2. Cu 2+ , Hg 2+ and Zn 2+ inhibited the soluble but they did not affect the lysosomal neuraminidase. Li + , Na + and K + , on the other hand, inhibited the lysosomal but not the soluble enzyme. Addition of Triton X-100 caused mild stimulation of the soluble neuraminidase and strong inhibition of the lysosomal enzyme. 3. 3. Ovine submaxillary glycoprotein is hydrolyzed by the lysosomal but not by the soluble neuraminidase. However, sialoglycopeptides, isolated from a pronase digest of ovine submaxillary glycoprotein, proved to be a good substrate for both enzymes. Neuramin-lactose sulfate was hydrolyzed at a higher rate than neuramin-lactose by both neuraminidases. Mixed brain gangliosides, the poorest of the substrates tested, were hydrolyzed by the soluble enzyme at a rate proportionally lower than that obtained with the lysosomal neuraminidase. 4. 4. Although a light and a heavy lysosome-rich fraction have been separated from rat mammary gland homogenates, no differences have been detected between the properties of the neuraminidase activity associated with these two fractions.

The invivo effect of benzamide and phenobarbital on liver enzymes: Poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.

Induction of rat liver microsomal and nuclear cytochrome p-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.

Effect of dietary butylated hydroxytoluene on nuclear envelope cytochrome p‐450 during the initiation and promotion stages of hepatocarcinogenesis

The anticarcinogenic effect of the dietary antioxidant butylated hydroxytoluene (BHT) correlates with a preservation of nuclear envelope (NE) cytochrome P-450 in rats undergoing chemically induced hepatocarcinogenesis. This effect of BHT on NE cytochrome P-450 was observed during both the initiation and the promotion stages of hepatocarcinogenesis. Complex interactions between the two different mechanisms of action of BHT (i.e., enzyme induction and antioxidant activity) may account for some of the differences between the patterns of response to BHT observed during initiation and promotion.

Prevention of 2-acetylaminofluorene-induced loss of nuclear envelope cytochrome P450 by the simultaneous administration of 3-methylcholanthrene

Rats fed a basal diet containing 0.05% (w/w) 2-acetylaminofluorene (AAF) for 3 weeks showed a 50% loss of hepatic nuclear envelope cytochrome P450, whereas microsomal P450 remained at control levels. A similar dietary treatment with 0.004% (w/w) 3-methylcholanthrene (MC) caused moderate losses (20-25%) of cytochrome P450 in both nuclear envelopes and microsomes. Administration of the basal diet supplemented with a mixture of AAF (0.05%) plus MC (0.004%) resulted in a preservation of control levels of nuclear envelope cytochrome P450 and a 30% elevation of microsomal P450. Immunoblot analysis revealed that AAF alone, or in concert with MC, induced comparable levels of the P450d form. Induction of cytochrome P450c by dietary MC was detected only when MC was fed together with AAF. As previously found for butylated hydroxytoluene (BHT), the protective effect of dietary MC against hepatocarcinogenesis in AAF-fed rats correlated with a preservation of nuclear envelope cytochrome P450 content and with the induction of cytochrome P450c.