Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.
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PMID:Metal ion activation of galactosyltransferase. 93 1

Avian embryonic sensory neurons from ED8 chick possess a trypsin-labile cell surface galactosyltransferase (GalTase) activity that glycosylates laminin in the presence of uridine 5' galactose (UDPgal). In a 4 h biological assay concentration dependent partial inhibition of neurite growth on laminin was observed in the presence of (i) alpha-lactalbumin, a specific inhibitor of the enzyme, (ii) N-acetylglucosamine (GlcNac), the appropriate acceptor substrate, or its polymer chitotriose, and (iii) UDPgal, the catalytic substrate. Prior exposure of substrate-immobilized laminin to glycosidase partially inhibited neurite growth. Alpha-lactalbumin did not influence cell adhesion at saturating concentrations for inhibition of neurite formation. Neurite growth on fibronectin was not inhibited by prior exposure to glycosidase or by alpha-lactalbumin, and fibronectin was not an appropriate substrate for glycosylation by the sensory neurons. These observations extend the catalogue of domains of laminin that subserve neurite growth, and define in functional terms a class of neuronal receptors that interact with lactosaminoglycan-type oligosaccharides of laminin.
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PMID:Neurite formation on laminin: effects of a galactosyltransferase on primary sensory neurons. 190 76

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.
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PMID:Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus. 218 69

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.
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PMID:Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes. 243 Jun 19

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.
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PMID:Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity. 247 71

During the last 15 years we have developed two biological systems, with whom it was possible to study the Ca++-dependent and the Ca++-independent adhesion on cellular level. In contrast to cells from other multicellular organisms, cells from the marine sponge Geodia cydonium are provided with Ca++-dependent adhesion mechanisms only. Two different mechanisms have been discovered by us, which were termed primary aggregation and secondary aggregation. In previous reports, we described that two macromolecules (aggregation factor [sAF] and aggregation receptor [AR] are involved in the secondary aggregation of sponge cells. The sAF was bound to a high-molecular-weight particle and was termed aggregation complex. The aggregation complex was shown to consist of two further functional subunits: UDP-glucuronosyltransferase and UDP-beta-D-galactosyltransferase. The AR with a molecular weight of approximately 17,000 was found to be a glycoprotein with D-glucuronic acid as the terminal sugar moiety. Data are presented from in vitro and in vivo experiments with the Geodia system, indicating that cell aggregation and cell separation are controlled first by alteration of the binding capacity of the aggregation receptor and second by an additional molecule (anti-aggregation receptor), which can decrease the interaction between the aggregation factor and the aggregation receptor. Recently we succeeded in the identification and isolation of the primary aggregation factor (pAF) from the same sponge species. This pAF is a glycoprotein that is firmly associated with the cell membrane. The Mr of the native pAF was 36,000; under denatured conditions three protein species were identified in the pAF preparation. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF. We were also able to dissociate the coral Eunicella cavolinii into single cells. These cells readily formed aggregates of a size of 2,100 micron during incubation in roller tubes: no aggregate formation was observed in non-rotating petri dishes. The formation of aggregates was not influenced by Ca++, urea or trypsin; it was also independent on temperature (4 degrees C to 30 degrees C) and pH (5.5-9.0). The intercellular material of the gorgonian contains a galactose-specific lectin, as determined by double diffusion experiments and haemagglutination inhibition experiments using a series of galacto-glycoconjugates. This lectin converted the aggregation-susceptible cells to aggregation-deficient cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The molecular mechanisms of the distinct calcium-dependent aggregation systems in marine sponges and corals. 286 62

The orientation of mannosidase II, an integral Golgi membrane protein involved in asparagine-linked oligosaccharide processing, has been examined in rat liver Golgi membranes. Previous studies on mannosidase II purified from Golgi membranes revealed an intact subunit of 124,000 daltons, as well as a catalytically active 110,000-dalton degradation product generated during purification (Moremen, K. W., and Touster, O. (1985) J. Biol. Chem. 260, 6654-6662). In Triton X-100 extracts of Golgi membranes, the intact enzyme was cleaved by a variety of proteases to generate degradation products similar to those observed previously. At appropriate concentrations, chymotrypsin, pronase, and proteinase K generated 110,000-dalton species, while trypsin and Staphylococcus aureus V8 protease generated 115,000-dalton forms. Cleavage by chymotrypsin under mild conditions (10 micrograms/ml, 10 min, 20 degrees C) resulted in a complete conversion to a catalytically active 110,000-dalton form of the enzyme which was extremely resistant to further degradation. Attempts to demonstrate these protease digestions in nonpermeabilized Golgi membranes were unsuccessful, a result suggesting that the protease-sensitive regions are not accessible on the external surface of the membrane. In Golgi membranes permeabilized by treatment with 0.5% saponin, mannosidase II could readily be cleaved to the 110,000-dalton form by digestion with chymotrypsin under conditions similar to those which result in a proteolytic inactivation of galactosyltransferase, a lumenal Golgi membrane marker. Although mannosidase II catalytic activity was not diminished by this chymotrypsin digestion, as much as 90% of the enzyme activity was converted to a nonsedimentable form. To examine the effect of the proteolytic cleavage on the partition behavior of the enzyme, control and chymotrypsin-treated Triton X-114 extracts of Golgi membranes were examined by phase separation at 35 degrees C. The undigested enzyme partitioned into the detergent phase consistent with its location as an integral Golgi membrane protein, while the 110,000-dalton chymotrypsin-digested enzyme partitioned almost exclusively into the aqueous phase in a manner characteristic of a soluble protein. These results suggest that mannosidase II catalytic activity resides in a proteolytically resistant, hydrophilic 110,000-dalton domain. Attachment of this catalytic domain to the lumenal face of Golgi membranes is achieved by a proteolytically sensitive linkage to a 14,000-dalton hydrophobic membrane anchoring domain.
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PMID:Topology of mannosidase II in rat liver Golgi membranes and release of the catalytic domain by selective proteolysis. 373 40

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2-4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophage-enriched, Fc receptor-bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4 degrees C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.
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PMID:Release of galactosyltransferase from peritoneal macrophages during acute inflammation. 393 May 17

A cell surface UDP-galactose:N-acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity-purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti-GT antibody resulted in its time-dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.
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PMID:Cell surface galactosyltransferase: immunochemical localization. 393 83

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP-sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.
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PMID:Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus. 678 76


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