Biomedical subjects

S Hirani

Publications and source records attributed to S Hirani.

At least 19 recordsLinked to original sources

PubMed1998

Experimental autoimmune encephalomyelitis induced with a combination of myelin basic protein and myelin oligodendrocyte glycoprotein is ameliorated by administration of a single myelin basic protein peptide.

Multiple sclerosis is an autoimmune disease of the central nervous system in which T cell reactivity to several myelin proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG), has been implicated in the perpetuation of the disease state. Experimental autoimmune encephalomyelitis (EAE) is used commonly as a model in which potential therapies for multiple sclerosis are evaluated. The ability of T cell epitope-containing peptides to down-regulate the disease course is well documented for both MBP- and proteolipid protein-induced EAE, and recently has been shown for MOG-induced EAE. In this study, we describe a novel EAE model, in which development of severe disease symptoms in (PL/J x SJL)F1 mice is dependent on reactivity to two different immunizing Ags, MBP and MOG. The disease is often fatal, with a relapsing/progressive course in survivors, and is more severe than would be predicted by immunization with either Ag alone. The MOG plus MBP disease can be treated postinduction with a combination of the MOG 41-60 peptide (identified as the major therapeutic MOG epitope for this strain) and the MBP Ac1-11[4Y] peptide. A significant treatment effect can also be obtained by administration of the MBP peptide alone, but this effect is strictly dose dependent. This MBP peptide does not treat the disease induced only with MOG. These results suggest that peptide immunotherapy can provide an effective means of mitigating disease in this model, even when the treatment is targeted to only one component epitope or one component protein Ag of a diverse autoimmune response.

Animals↗

PubMed1996

IL-4 production by allergen-stimulated primary cultures: identification of basophils as the major IL-4-producing cell type.

As a potent inducing agent for IgE production and differentiation factor for allergen-specific Th2 cells, IL-4 is a key regulatory cytokine both in the pathogenesis of allergic disease and in the ongoing allergic response. The assay of in vitro IL-4 production has often been used to compare the allergen responses of T cells isolated from atopic and non-atopic subjects. Because peripheral blood basophils also have the capacity to respond to specific allergen by producing IL-4, we investigated the relative contribution of these two cell types to IL-4 production in allergen-stimulated primary cultures. Among unfractionated peripheral blood mononuclear cells (PBMC), the major producers of detectable IL-4 in primary in vitro cultures were found to be basophils based on: (i) an allergen dose-response corresponding closely to that required for basophil histamine release and lower than that required for T cell activation; (ii) a rapid time course for IL-4 production (detectable at 3 h), inconsistent with the typical activation requirements of fresh T cells; (iii) the production of comparable levels of IL-4 in cultures stimulated with allergen or anti-IgE; and (iv) the complete loss of detectable IL-4 production following specific depletion of basophils from PBMC. The T cells in these cultures were functionally able to produce IL-4, as demonstrated by mitogen activation of basophil-depleted PBMC. These findings demonstrate that although IL-4 production in primary in vitro cultures can be used as a sensitive indicator of allergen responsiveness, the accurate interpretation of this result requires identification of the responding cell type. Furthermore, these findings raise the possibility that basophil production of IL-4 early in the course of allergen stimulation may shape subsequent T cell responses both in vivo and in vitro.

Allergens↗

PubMed1994

1H NMR studies on an Asn-linked glycopeptide. GlcNAc-1 C2-N2 bond is rigid in H2O.

The conformation of an Asn-linked glycopeptide in H2O was studied by two-dimensional 1H NMR. Nonexchangeable proton and exchangeable amide (NH) proton resonances were assigned for the hen ovomucoid glycopeptide 1, Ser-Ile-Glu-Phe-Gly-Thr-Asn Ile-Ser-Lys, with pentasaccharide Man alpha 1-3 (Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc beta 1-NH attached to the Asn7 gamma-carboxamide. The pentasaccharide increases the local correlation times of amino acid residues near the N-glycosylation site. Nuclear Overhauser effect (NOE) measurements on 1 and the corresponding Man3-GlcNAc2 pentasaccharide 3 show that the attached peptide does not perturb O-glycoside conformation. Sequential dNN (i, i + 1) NOEs in the Thr6-Ser9 region indicate populations of folded structure near the N-glycosylation site of both glycopeptide 1 and aglycosyl peptide 2. However, the Man3GlcNAc2 pentasaccharide does not dramatically affect the average conformation of either the peptide backbone or the Asn7 side chain. GlcNAc NH protons were studied at pH 3.0; and NOE and 3JNH data were used to constrain the glycopeptide's GlcNAc-1 side chain dihedral angle (tau) (C1-C2-N2-C7(Ac)). The glycopeptide's core GlcNAc-1 C2-N2 side chain bond is not flexible in H2O. A strong GlcNAc-1 NH2-H3 NOE, a medium strength NH2-H1 NOE, and a weak NH2-H2 interaction suggest that GlcNAc-1 has a rigid C2-N2 bond, with tau between 95 and 115 degrees. No evidence was found for intramolecular hydrogen bonds restricting this C2 side chain torsion. It may be that GlcNAc-1's rigid planar N-glycosidic linkage limits the conformational space available to the adjacent C2 acetamido side chain.

Acetylglucosamine↗

PubMed1992

Substrate specificity of human liver neutral alpha-mannosidase.

The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol.

Carbohydrate Conformation↗

PubMed1991

Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.

A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.

Animals↗

PubMed1990

Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli.

The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.

Amidohydrolases↗

PubMed1987

Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis.

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.

Borohydrides↗

PubMed1987

A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex.

We have identified a vesicle fraction that contains alpha 1-antitrypsin and other human HepG2 hepatoma secretory proteins en route from the rough endoplasmic reticulum (RER) to the cis face of the Golgi complex. [35S]Methionine pulse-labeled cells were chased for various periods of time, and then a postnuclear supernatant fraction was resolved on a shallow sucrose-D2O gradient. This intermediate fraction has a density lighter than RER or Golgi vesicles. Most alpha 1-antitrypsin in this fraction (P1) bears N-linked oligosaccharides of composition similar to that of alpha 1-antitrypsin within the RER; mainly Man8GlcNac2 with lesser amounts of Man7GlcNac2 and Man9GlcNac2; this suggests that the protein has not yet reacted with alpha-mannosidase-I on the cis face of the Golgi complex. This light vesicle species is the first post-ER fraction to be filled by labeled alpha 1-antitrypsin after a short chase, and newly made secretory proteins enter this compartment in proportion to their rate of exit from the RER and their rate of secretion from the cells: alpha 1-antitrypsin and albumin faster than preC3 and alpha 1-antichymotrypsin, faster, in turn, then transferrin. Deoxynojirimycin, a drug that blocks removal of glucose residues from alpha 1-antitrypsin in the RER and blocks its intracellular maturation, also blocks its appearance in this intermediate compartment. Upon further chase of the cells, we detect sequential maturation of alpha 1-antitrypsin to two other intracellular forms: first, P2, a form that has the same gel mobility as P1 but that bears an endoglycosidase H-resistant oligosaccharide and is found in a compartment--probably the medial Golgi complex--of density higher than that of the intermediate that contains P1; and second, the mature sialylated form of alpha 1-antitrypsin.

1-Deoxynojirimycin↗

PubMed1987

In vivo evaluation of hydroxypyridone iron chelators in a mouse model.

The 59Fe excretion caused by a range of bidentate N-substituted [R group = methyl (CP20), ethyl (CP21), propyl (CP22), isopropyl (CP23), butyl (CP24) or hexyl (CP25)] 3-hydroxypyrid-4-one chelators in iron-overloaded mice is presented. All the compounds cause significant iron excretion when given intraperitoneally, but that the most hydrophobic compounds, CP24 and CP25, were toxic except at low doses. The excretion caused by CP21, CP22 and CP23 were significantly greater than that caused by CP20 and slightly larger than that caused by an equivalent dose of desferrioxamine. These compounds (CP20 through CP24) also caused significant excretion of 59Fe when administered orally. Compounds CP21, CP22 and CP24 were significantly more active than compounds CP20 and CP23. It is concluded that the N-ethyl or N-propyl 3-hydroxypyrid-4-ones are the most promising compounds for clinical application. Preliminary experiments using a hexadentate pyrid-2-one, CP130, show that this causes significant 59Fe excretion both when given intraperitoneally or orally.

Administration, Oral↗

PubMed1986

Structural analysis of the asparagine-linked oligosaccharides of human complement component C3.

The asparagine-linked oligosaccharides of human C3 were characterized. The C3 oligosaccharides were released by endo-beta-N-acetylglucosaminidase H and were analysed by lectin affinity chromatography and h.p.l.c. The released oligosaccharides bound tightly to concanavalin A-Sepharose and were not retained by agarose-bound wheat-germ agglutinin, indicating that they were only of high-mannose type. Two major oligosaccharide structures were separated from both the alpha- and beta-chains of C3 by h.p.l.c. on Micropak AX-5, calibrated with high-mannose-type oligosaccharides of known structures. The oligosaccharide structures on the alpha-chain have the compositions (Man)9(GlcNAc)2-Asn and (Man)8 (GlcNAc)2-Asn, and those on the beta-chain have the compositions (Man)6(GlcNAc)2-Asn and (Man)5(GlcNAc)2-Asn.

Asparagine↗

PubMed1985

Changes in human plasma alpha-D-mannosidase activity during pregnancy.

There are significant and progressive increases in plasma acidic (pH optimum 4.2) and intermediate (pH optimum 5.6) alpha-mannosidase during pregnancy. The acidic alpha-mannosidase in plasma from pregnant women binds to concanavalin A and has the same apparent molecular weight as the acidic alpha-mannosidase in control plasma. The 2-3-fold increase in acidic alpha-mannosidase in pregnancy is due to an increase in the most negatively charged form of acidic alpha-mannosidase, B2, which is slightly more negatively charged than its counterpart in the control plasma. The intermediate alpha-mannosidase, which increases by approximately 50% during pregnancy, can be resolved by a combination of chromatography on concanavalin A and gel filtration into the same forms found in control plasma.

Chromatography, Affinity↗

PubMed1985

Leukocyte complement: interleukin-like properties of factor Bb.

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".

Complement C3↗

PubMed1985

Complement activation in type 1 human diabetes.

Complement activation was quantitated in serum and plasma of diabetic and normal subjects by sensitive competitive equilibrium radioimmunoassays (RIA) for C3a, C4a, C5a, Factor B, and a newly described C5 neoantigen (termed C5 activation antigen, and abbreviated C5-AA) in a stable 54-kDa fragment of C5. Plasma C3a levels were significantly elevated in 8 of 16 patients with newly diagnosed Type 1 diabetes (P less than 0.0005) with the mean C3a concentration for these patients being more than 10-times greater than the mean value of normal controls. C4a levels were also elevated in 2 of these patients (P less than 0.02), but C5a levels, although higher than normal, were not significantly increased. In contrast, the levels of C5-AA in the serum of all patients (11/11) with chronic Type 1 diabetes were significantly higher than in control Type 2 patients (noninsulin-dependent diabetes) (P less than 0.0005) and 4 of 7 patients with new onset insulin-dependent diabetes mellitus also had significantly higher levels of C5-AA than the Type 2 patients (P less than 0.01). The levels of Factor B in the serum of 5 of 9 patients with new onset diabetes were significantly higher than normal (P less than 0.0025). Five recent onset Type 1 diabetes patients were evaluated longitudinally for C3a, C4a, and C5a: in 3 the levels of C3a were elevated during new onset disease decreasing into the normal range during remission; in 2 of these patients C4a was also significantly elevated and the levels decreased during remission; and in 3 patients the levels of C5a were not significantly elevated but they decreased during remission. Purified human complement proteins and complement hemolytic assays were used to measure complement activation in serum during incubation with rat pancreatic islet cells. With diluted normal human serum, less than 20% of C3 or Factor B were consumed during 30 min at 37 degrees C, while with new onset Type 1 diabetic patient sera up to 90% of C3 and Factor B were consumed in 5/6 sera and 4/6 sera, respectively. These findings suggest (a) that complement activation fragments C3a, C4a, and C5a are generated in vivo in new onset Type 1 diabetes; (b) that both the classical and the alternative complement pathways may be activated; and (c) that this may result in a measurable activation of C5 generating biologically and immunologically active C5a and other C5 activation fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗

PubMed1985

Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement.

The C3d domain of C3 contains the site that binds to the C3d receptor (CR2) which is expressed on B lymphocytes. It also contains a neoantigenic determinant that is recognized by monoclonal antibody (mAb) 130 and is expressed when C3b is cleaved to iC3b and subsequently to C3dg or C3d. mAb 130 inhibits the binding of C3d to CR2. In this study, the locations of the CR2-binding site and of the neoantigen recognized by mAb 130 within the C3d domain were investigated. Treatment of human C3d with CNBr generated two major fragments with Mrs of 12,500 and 8600. Binding studies showed that only the Mr 8600 fragment was capable of binding to both CR2 and mAb 130. Amino-terminal sequence analysis of the Mr 8600 fragment and comparison with the amino acid sequence derived from human C3 cDNA [de Bruijn, M. H. L. & Fey, G. H. (1985) Proc. Natl. Acad. Sci. USA 82, 708-712] placed it between residues 1199 and 1274 of the C3 sequence. Several peptides were synthesized according to the derived C3 sequence of amino acid residues 1209-1236. Based on their differential binding to CR2 and mAb 130, we localized the CR2-binding site and mAb 130 neoantigenic site, respectively, to residues 1227-1232 and 1217-1232 of the C3 sequence.

Amino Acid Sequence↗

PubMed1985

Localization of the conglutinin binding site on the third component of human complement.

The binding site on the human third complement component for bovine conglutinin has been located. C3 fragments were purified to homogeneity by preparative SDS-polyacrylamide-gel electrophoresis. Only the N-terminal 27,000 dalton (Da) fragment of the alpha'-chain and the beta-chain were found to be glycosylated, and the carbohydrate was susceptible to endo-beta-N-acetylglucosaminidase H. This finding indicates that only high mannose or hybrid-type oligosaccharide chains are present on the C3 molecule. Binding to conglutinin was determined by an enzyme-linked immunosorbent assay and occurred with C3b, iC3b, C3c, the alpha-chain, and the 27,000 Da fragment of the alpha'-chain, but not with C3d or the C-terminal 40,000 Da fragment of the alpha'-chain. The beta-chain displayed very weak interaction. Binding to conglutinin could be inhibited by EDTA, N-acetylglucosamine, and to a lesser degree by mannose. Enzymatic removal of the carbohydrate from the C3 molecule abolished binding to conglutinin. It is concluded that bovine conglutinin binds to the carbohydrate moiety located on the N-terminal 27,000 Da polypeptide of the alpha-chain.

Acetylglucosamine↗

PubMed1982

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties.

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.

Carbohydrates↗

PubMed1982

A study of highly purified mucolipidosis III urinary N-acetyl-beta-D-hexosaminidase B.

Highly purified N-acetyl-beta-D-hexosaminidase B from normal urine and urine of a patient with mucolipidosis III was used to determine whether it has undergone any of the alterations associated with this genetic defect. Examination by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that both the enzyme preparations contained protein components with apparent Mr values of 55 000 and 28 000. No differences in the binding and apparent KI (50%) to concanavalin A of the normal and mucolipidosis III enzymes were detected. However, the patient's N-acetyl-beta-D-hexosaminidase B had a slightly greater affinity for the lectin from Ricinus communis than did the normal enzyme. Two-dimensional tryptic peptide maps of the corresponding normal and the patient's N-acetyl-beta-D-hexosaminidase B subunits showed considerable homology. These results indicate that N-acetyl-beta-D-hexosaminidase b does not undergo the significant carbohydrate alterations characteristic of other acid hydrolases in mucolipidosis III.

Concanavalin A↗

PubMed1980

Biochemical studies on a case of feline mannosidosis.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.

Animals↗