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Biomedical subjects

D Fabbro

Publications and source records attributed to D Fabbro.

At least 145 records · Page 8Linked to original sources
PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗
PubMed1986

Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship.

Quantitative polyacrylamide gel electrophoresis analysis of Ca2+, phospholipid-dependent protein kinase (PKC) of human mammary tumor cell lines (MCF-7, ZR-75, T-47-D, MDA-MB-231, BT-20, and HBL-100) revealed that 80% of the total cellular PKC resided in the cytosol. The tumor cells with no detectable levels of estrogen receptors (MDA-MB-231, HBL-100, and BT-20 cells) exhibited significantly larger (P less than 0.001) cytosolic PKC activities than those cells that contained estrogen receptors (MCF-7, T-47-D, and ZR-75 cells). In addition, in estrogen receptor-negative cell lines, relatively high levels of specific low-affinity (apparent Kd = 700 pM) epidermal growth factor (EGF) binding activities were found as compared with estrogen receptor-positive cells with significantly (P less than 0.001) lower levels of specific high-affinity (apparent Kd = 90 pM) EGT binding. A significant positive correlation (P less than 0.01) was observed between the number of EGF receptor (Rs = 0.50) and/or the EGF receptor dissociation constants (Rs = 0.78) with the cytosolic PKC activity levels. These data indicate that, in human breast cancer cells, a positive relationship may exist between PKC activity, estrogen, and EGF receptors.

Breast Neoplasms↗
PubMed1985

The cytosolic phorboid receptor correlates with hormone dependency in six mammary carcinoma cell lines.

Potent, structurally different tumor promoters inhibited growth of 6 human mammary carcinoma cell lines (ROOS et al, PNAS in press). This growth inhibition was investigated by measuring the phorboid receptor binding using [3H] PDBu (4 beta-phorbol 12, 13 dibutyrate). Specific, high affinity receptors were found in all six cell lines. [3H] PDBu binding affinities were higher in the cytosolic fractions than in the corresponding intact cells (K alpha = app. 1nM vs K alpha = app. 15nM). The hormone-independent cell lines (BT-20, HBL-100 and MDA-MB-231) exhibited significantly higher levels of cytosolic [3H] PDBu receptors than the hormone-dependent cells (MCF-7, T-47-D and ZR-75-1). The subcellular distribution of the [3H] PDBu binding correlated well with the distribution of the protein kinase C activity (r = 0.95).

Breast Neoplasms↗
PubMed1985

Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis.

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).

Animals↗
PubMed1985

cAMP-dependent protein kinases of rat pituitary GH3 cells.

Two isoenzymes (type I and type II) of cAMP-dependent protein kinases were found in GH3 cytosol, isozyme type II activity being the predominant form (approximately 90%). Photoaffinity labeling of GH3 cell extracts with 8-N3-[32P]cAMP revealed three cAMP-binding proteins exhibiting molecular weights of 53,000, 51,000 and 48,000, respectively. The latter represents the regulatory subunit of type I isoenzyme whereas the 53,000 and 51,000 cAMP-binding proteins correspond to two different molecular forms of the type II isoenzyme regulatory subunit which are phosphorylated by a cAMP-dependent mechanism.

Animals↗
PubMed1984

Lysosomal beta-glucosidase of rat liver.

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic beta-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-beta-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Phosphorylation of cAMP-dependent protein kinases in normal and abnormal human sperm.

The molecular characterization and distribution of cAMP-dependent protein kinases (cAPK) of normal and pathological (reduced progressive motility or altered morphology) human semen were investigated. Photoaffinity labeling with 8-N3-[32P]cAMP of human sperm cytosols revealed four specific cAMP-binding proteins (MW: 52 kD, 47 kD, 42 kD, and 37 kD) with the following quantitative distribution: 65% (47 kD), 30% (52 kD), and less than 5% (42/37 kD). In contrast, the seminal plasma of the respective semen samples incorporated the 8-azido cAMP photolabel predominantly into the 37 kD protein (greater than 50%). The cAMP-binding proteins with MW of 52 kD and 47 kD corresponded to the regulatory subunits R II and R I of the respective cAPK isoenzymes I and II. Neither qualitative nor quantitative differences could be detected in the distribution between seminal plasma and sperm as well as in the molecular properties of RI (47 kD), R II (52 kD), and of their proteolytic products (42 kD and 37 kD) between normal and pathological human semen. DEAE-cellulose chromatography revealed that type II isozyme was the predominant form (80% of the total cAMP-dependent protein kinase activity of sperm); the protein kinase isozyme pattern was similar in normal and pathological sperm. DEAE-cellulose chromatography in combination with photoaffinity labeling (8-N3-[32P]cAMP) resolved the kinase activity into type I and type II isoenzymes and into the corresponding subunits R I (47 kD) and R II (52 kD). The excessive amount of R I (47 kD) found by photoaffinity labeling in comparison to type I holoenzyme after DEAE-cellulose chromatography is due to a higher binding affinity of R I (5 nM) for the 8-N3[32P]cAMP as compared with that of R II (50 nM). In addition, endogenous phosphorylation of soluble sperm proteins revealed that the R II (52 kD) was present only as the phosphoform of R II.

Adolescent↗
PubMed1983

Cyclic AMP-dependent protein kinases in semen from men with subnormal sperm motility or morphology.

Photoaffinity labelling by 8-N3-/32P/cAMP of human sperm homogenates with normal pathology and normal progressive motility of the gametes (N) revealed the presence of 2 specific cAMP-binding activities with MW 47 000 (R-I) and 52 000 (R-II). Similar amounts of R-I and R-II were found in sperm homogenates obtained from semen samples with either reduced progressive motility (A50: 30-50% progressive motility and A30: less than 30% progressive motility) or abnormal morphology of the gametes (T: 5-30% normal heads). In contrast, the seminal plasma of the respective semen samples of N, A50, A30 and T incorporated the 8-azido cAMP photolabel in similar quantities into 2 additional cAMP-binding proteins with MW 42 000 and 37 000. Quantitative analysis of the photoaffinity labelling of the seminal plasma obtained from normal (N) or subnormal semen (A50, A30, T) showed that cAMP was predominantly bound to the 37 000 protein. Furthermore, the cAMP-dependent protein kinase and cAMP-binding activities of sperm homogenates were quantitatively comparable within the 4 groups. These results provide the first evidence that neither quantitative nor qualitative differences in the molecular and/or enzymatic properties of cAMP-dependent protein kinases can be detected between normal human sperm (N) and those with either reduced progressive motility (A50, A30) or abnormal morphology (T).

Adolescent↗
PubMed1982

Gaucher disease: a membranous enzymopathy.

Inhibitors and activators of acid beta-glucosidase activity were used as probes to characterize the components of the active site of the membrane bound enzyme, acid beta-glucosidase. Three components of the active site were defined: (1) a catalytic site, (2) an aglycon binding site, and (3) a hydrophobic binding site (Fig. 5A). Similar studies on the residual acid beta-glucosidase in homozygotes with Type 1 Ashkenazi Gaucher disease suggested that this enzyme's hydrophobic site was more hydrophilic than that of the normal enzyme. The defect in this membranous enzymopathy could have resulted from a single base substitution in the structural gene leading to the insertion of a more hydrophilic amino acid in the hydrophobic domain of the gene product. Alternatively, a base substitution which altered the conformation of the enzyme could render the hydrophobic site more hydrophilic. The following consequences of such a mutation would be expected. The mutation would not affect substrate binding to the catalytic site, since the formation of the enzyme-substrate complex (ie, the Km) would not be altered. If the HS site became more hydrophilic, its efficiency for removing the product would be reduced, resulting in a lower substrate turnover (ie, a "Vmax mutation"). Consequently, the binding of glucosyl psychosine, taurocholate, and phosphatidyl serine to the hydrophobic site would be less efficient, resulting in a greater alpha Ki value. Finally, the binding of taurocholate to the HS would be reduced, and this lipid's enhancement of acid beta-glucosidase inhibition by OBG also would be decreased. Since these results are based on kinetic data, confirmation of the hypothesis will require the preparation of homogenous beta-glucosidase from normal and Type 1 Ashkenazi Gaucher sources for amino acid sequencing of the peptides containing the catalytic site as well as the other components of the active site. Such peptides might be identified by their ability to bind radiolabeled inhibitors and/or activating compounds.

Binding Sites↗
PubMed1981

Nuclear protein kinases of GH3 cells.

Three distinct cAMP-independent nuclear protein kinase activities (PCV, PC-II and PC-III) have been separated by phosphocellulose chromatography of GH3 nuclear NaCl extracts. The kinase activities exhibit distinct substrate specificities and preference for either the basic substrates protamine sulfate and histones H1 and H3 (kinase activity PCV) or the acidic protein substrates casein (PC-II) and phosvitin (PC-III). The three nuclear protein kinases further differ in their estimated molecular weights, apparent Km values and in their sensitivity toward NaCl. All three kinases require Mg2+ for optimal activity.

Animals↗
PubMed1980

Progesterone and estrogen receptors in GH3 cells.

GH3 cells are shown to contain cytosolic progesterone receptor in a mean concentration of 270 fmol/mg protein with a dissociation constant (K(D)) for promegestone of 3 times 10(-9) M (4 degrees C). Estrogen receptor (K(D) = 1.8 times 10(-10) M) is demonstrated in cytosol (121 fmol/mg protein) as well as in 0.4 M KCl extracts (89 fmol/mg protein) of crude nuclear fractions. No progesterone receptor was detectable in the nuclear fraction. Both receptors are characterized by isoelectric focussing and by competition experiments. Addition of 10(-8) M estradiol to the growth medium increases progesterone receptor levels up to five, suggesting that the progesterone receptor is under estrogenic control.

Animals↗
PubMedDate not supplied

Inhibitors of protein kinases: CGP 41251, a protein kinase inhibitor with potential as an anticancer agent.

CGP 41251 was originally identified as an inhibitor of protein kinase C (PKC), inhibiting mainly the conventional PKC subtypes, and subsequently shown to inhibit the vascular endothelial growth factor (VEGF) receptor kinase insert domain-containing receptor, which is involved in angiogenesis. CGP 41251 inhibits reversibly intracellular PKC activity, induction of c-fos and the corresponding activation of the mitogen-activated protein kinase induced by either tumor promoting phorbol esters, platelet-derived growth factor, or basic fibroblast growth factor, but not by the epidermal growth factor. CGP 41251 inhibited the ligand-induced autophosphorylation of the receptors for platelet-derived growth factor, stem cell factor, and VEGF (kinase insert domain-containing receptor) that correlated with the inhibition of the mitogen-activated protein kinase activation, but did not affect the ligand-induced autophosphorylation of the receptors for insulin, insulin-like growth factor-I, or epidermal growth factor. CGP 41251 showed broad antiproliferative activity against various tumor and normal cell lines in vitro, and is able to reverse the p-glycoprotein-mediated multidrug resistance of tumor cells in vitro. CGP 41251 showed in vivo antitumor activity as single agent and inhibited angiogenesis in vivo. Thus, CGP 41251 may suppress tumor growth by inhibiting tumor angiogenesis (via its effects on the VEGF receptor tyrosine kinases) in addition to directly inhibiting tumor cell proliferation (via its effects on PKCs).

Antineoplastic Agents↗