Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 10(-5) M). "High-uptake" fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand.     

Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D -glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.     

Inhibition of fungal growth by plant chitinases andβ-1,3-glucanases

Summary Plant chitinases and -1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and -1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.Abbreviations WGA-FITC wheat germ agglutinin labelled with fluorescein isothiocyanate - ConA-FITC concanavalin A labelled with fluorescein isothiocyanate - PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy     

Biochemical Cytology of Trichomonad Flagellates. II. Subcellular Distribution of Oxidoreductases and Hydrolases in Monocercomonas sp.*

A primitive trichomonad, Monocercomonas sp. (strain NS-1:PRR) from Natrix sipedon, was grown axenically in Diamond's medium. Activity of NADH oxidase, malate dehydrogenase, malate dehydrogenase (decarboxylating) and of the anaerobic enzymes, pyruvate synthase and hydrogenase as well as of several hydrolases was demonstrated in homogenates. The subcellular distribution of these activities was studied by means of analytical differential and isopycnic centrifugation of homogenates prepared in 0.25 M sucrose. NADH oxidase and malate dehydrogenase are in the nonsedimentable part of the cytoplasm. Malate dehydrogenase (decarboxylating), pyruvate synthase, and hydrogenase are associated with a large particle which equilibrates at density 1.22. In its properties, this particle corresponds to the microbody-like hydrogenosomes of Tritrichomonas foetus. The 5 hydrolases studied are associated with at least 2 different particle populations: a large particle population equilibrating at densities from 1.10 to 1.16 is the exclusive location of 3 enzymes (β-galactosidase, protease and β-N-acetylglucosaminidase), 2 of which have a pH optimum close to neutrality. These particles contain part of the acid phosphatase and β-glucuronidase. Another part of these 2 enzymes is associated with a separate population of smaller granules with equilibrium densities of 1.16 to 1.18. The 2 types of hydrolase-carrying particles are also biochemically very similar to their counterparts in T. foetus.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Castor-bean acid lipase catalysed hydrolysis of triacylglycerols does not involve C-2 → C-1,3 transacylation

The hydrolysis of a series of triacylglycerol analogs catalysed by castor-bean acid lipase was studied at 30° and pH 4.20. Iso-propyl esters underwent lipolysis, thus refuting the mechanistic proposition that hydrolysis at C-2 in triacylglycerols occurs via a slow transfer of the acyl moiety from C-2 to either C-1 or C-3, followed by enzymic hydrolytic action.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

Synthesis of the tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp recognized by calreticulin/calnexin

The title compound as its methyl glycoside was efficiently synthesized using a block synthesis approach. Halide-assisted glycosidations between 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl iodide and ethyl 2-O-acetyl-4,6-di-O-benzyl-1-thio-alpha-D-mannopyranoside using triphenylphosphine oxide as promoter yielded, with complete alpha-selectivity, a disaccharide building block in high yield. The perbenzylated derivative of this proved to be an excellent donor affording 88% of the protected target tetrasaccharide in an NIS/AgOTf-promoted coupling to a known methyl dimannoside acceptor. Deprotection through catalytic hydrogenolysis then gave the target compound in 47% overall yield.     

Entamoeba histolytica: biochemical and molecular insights into the activities within microsomal fractions

One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.     

A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity

Glycosyl hydrolases are a vast group of enzymes that share a common topology at their active site with two acidic residues that are responsible for activity. In spite of their similarity, they exhibit a wide range of pH optima that must depend on other factors. Using structural and mechanistic knowledge about glycosyl hydrolases from families 7, 10, and 16, we have formulated a new mathematical model that can include not only the ionization behavior of the catalytic residues but also as many ionizable residues as desired in the active site. In addition, the model can incorporate electrostatic influences via acid dissociation equilibrium constants and chemical relationships such as hydrogen bonds. The results of the simulations indicate a clear shift in the pH dependence of activity for the enzymes only when a close interrelation (hydrogen bond) between the catalytic and auxiliary residues in the active site is taken into account. This explains the observations from mutagenesis studies that show this type of shift and cannot be explained by a purely electrostatic interaction theory. Moreover, the presence of the kind of chemical interaction proposed could provide stabilization of the activity in the presence of environmental, structural, pH and electrostatic variations. These findings and the implications for the design of new mutagenesis strategies are discussed. The results suggest a way to modify, via site-directed mutagenesis, the acid dissociation of one of the catalytic residues in the active site independently of the other, which could have clear advantages over the purely electrostatic modifications that usually affect both residues simultaneously.