Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

血红细胞超氧化物歧化酶(SOD)制备工艺的研究初报

本文报告了本实验室设计的由血红细胞自溶液60℃热变性, 乙醇——氯仿法除血红蛋白,旋转蒸发法减压浓缩抽去氯仿、乙醇,硫酸铵分级盐析法沉降SOD,Sepbadex G-75层析提纯SOD等步骤构成的一条成本低、设计合理、简便实用的分离纯化SOD的工艺路线。     

Solubilisation et caractérisation d'une glycoprotéine : fucosyl-transférase cérébrale

1. A glycoprotein: fucosyl-transferase activity was demonstrated in sheep cerebral cortex, using desialylated fetuin as exogenous acceptor and detergent Trition X 100 for solubilization.

2. Addition of Triton X 100 to the membrane suspension gave first an activation then a solubilization of the cerebral fucosyl-transferase.

3. Hydrophobic chromatography was investigated for purification of the enzyme. Binding was effective using alkyl-agarose chromatographic columns with two or more than two atoms of carbon, but elution was only possible with ethyl and butyl-agarose.

4. Combination of subcellular fractionation and hydrophobic chromatography on ethylagarose led to a 30 fold purification.

5. After ethyl-agarose chromatography, some properties of fucosyl-transferase were studied: the optimal temperature was 25°C. The optimum pH was about neutrality. Light activation was observed with Mn²⁺ concentration below 1 mM.

6. Homogeneity was tested using Ultrogel chromatography, polyacrylamide gel electrophoresis and ultracentrifugation.

7. It was concluded that ethyl-agarose hydrophobic chromatography easily bind a few solubilized proteins (about 20 per cent of the ST supernatant). When elution was performed, these proteins, including fucosyl-transferase, were released from ethyl-agarose columns as a stable aggregate, only dissociated with lower ionic strength.

Ethyl-agarose fraction (eluted with KCl 120 mM) showed homogeneity:

— with Ultrogel AcA 22 chromatography (Mw = 300.000).

— with polyacrylamide gel electrophoresis without S.D.S.

— with the analytical ultracentrifugo giving Mw = 280.000; S₂₀ = 10.

But after dialysis overnight against a buffer without KCl, ultracentrifugation technics showed no homogeneity. Futhermore, SDS gel electrophoresis gave more than four bands.     

Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage.

Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.     

Affinity of Ionic Species of Nucleoside Transport Protein Inhibitors

Abstract

A class of very potent nucleoside transport inhibitors is present in two molecular forms around physiological pH. We investigated whether the monoprotonated or the unionized species of these molecules binds to this camer protein with higher affinity.     

THE EFFECTS OF INJURY PREVENTION WARM-UP PROGRAMMES ON KNEE STRENGTH IN MALE SOCCER PLAYERS

The study investigates the effects of the 11+ and HarmoKnee injury prevention programmes on knee strength in male soccer players. Under-21-year-old players (n=36) were divided equally into: the 11+, HarmoKnee and control groups. The programmes were performed for 24 sessions (20-25 min each). The hamstrings and quadriceps strength were measured bilaterally at 60°·s^-1, 180°·s^-1 and 300°·s^-1. The concentric quadriceps peak torque (PT) of the 11+ increased by 27.7% at 300°·s^-1 in the dominant leg (p<0.05). The concentric quadriceps PT of HarmoKnee increased by 36.6%, 36.2% and 28% in the dominant leg, and by 31.3%, 31.7% and 20.05% at 60°·s^-1, 180°·s^-1 and 300°·s^-1 in the non-dominant leg respectively. In the 11+ group the concentric hamstring PT increased by 22%, 21.4% and 22.1% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, respectively in the dominant leg, and by 22.3%, and 15.7% at 60°·s^-1 and 180°·s^-1, in the non-dominant leg. In the HarmoKnee group the hamstrings in the dominant leg showed an increase in PT by 32.5%, 31.3% and 14.3% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, and in the non-dominant leg hamstrings PT increased by 21.1% and 19.3% at 60°·s^-1 and 180°·s^-1 respectively. The concentric hamstrings strength was significantly different between the 11+ and control groups in the dominant (p=0.01) and non-dominant legs (p=0.02). The HarmoKnee programme enhanced the concentric strength of quadriceps. The 11+ and HarmoKnee programmes are useful warm-up protocols for improving concentric hamstring strength in young professional male soccer players. The 11+ programme is more advantageous for its greater concentric hamstring strength improvement compared to the HarmoKnee programme.