beta-N-acetylhexosaminidase isoenzymes during chick embryo development

1. Two forms (I and II) with acidic pH optima and a neutral form of beta-hexosaminidase has been separated by DEAE-cellulose chromatography and characterized in skin and lung of 7, 9, 11, 14 day chick embryos and 1 day old chicken. 2. Forms I and II are similar to hexosaminidase A and B for their behaviour on DEAE-cellulose chromatography, Concanavalin A-Sepharose column and thermal stability. 3. Neutral form has a neutral pH optimum and higher molecular weight and a more acidic I. P. than forms I and II, a low beta-N-acetylgalactosaminidase activity and it is not bound by a Concanavalin A-Sepharose column and in that resemble hexosaminidase C and/or other neutral hexosaminidases. 4. We have found differences in the percentage of neutral form and in the specific activities of the extracts in the skin in different stages of development. 5. No significant differences were observed in the lung.     

Irreversible inhibition of hexosaminidase C by medium-chain monocarboxylic acids and Triton X-100

The neutral beta-N-acetylhexosaminidase (hexosaminidase C) from human brain was partially purified (separated from lysosomal beta-N-acetylhexosaminidases by chromatography on a Con A-Sepharose column). Hexosaminidase C was inhibited by medium-chain fatty acids (monocarboxylic acids with chain-length between C6 and C9), whereas shorter-chain monocarboxylic acids showed no inhibitory effect. Studies on the inhibition mechanism showed an irreversible and pH-dependent inhibition which progresses with time and which is not reversed by the removal of fatty acids (by Bio-Beads SM-2). Similar inhibitory effects were also obtained using Triton X-100 (but not with homologous alkylamines). These results suggest that the hexosaminidase C inactivation is related to the hydrophobic properties of the inhibitor which acts as a denaturing agent mainly at acidic pH. The possibility has been discussed that this inactivation effect of monocarboxylic acid on hexosaminidase C could constitute a molecular model of the toxicity of medium-chain-length fatty acids.     

Mannosidosis in Angus cattle. The enzymic defect

Normal calf alpha-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of alpha-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn(2+), Co(2+) and Mn(2+). An acidic alpha-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic alpha-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5-5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic alpha-mannosidase activity.     

Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral beta-N-acetylhexosamindases

The bulk of rat brain neutral beta-N-acetylhexosaminidases (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) were present in the cytosol fraction. They were not bound by concanavalin A-Sepharose while the acid beta-N-acetylhexosaminidases were all bound. The neutral beta-N-acetylgalactosaminidase had a pH optimum of 5.2 and Km of 0.57 mM, while the neutral beta-N-acetylgalactosaminidase had the highest reaction rate at lost more than 90% of the activity in 30 min at 50 degrees C. The galactosaminidase pH 6.0 with a Km of 0.12 mM. No divalent ions activated either of the enzymes. The galactosaminidase was heat-stable and lost only 10--20% of its activity after 3 h at 50 degrees C. The neutral glucosaminidase was inhibited by free N-acetylglucosamine but not by N-acetylgalactosamine. The reverse was found for the neutral beta-galactosaminidase. Two enzymes were separated almost completely by hydroxyapatite chromatography. Heat stability of the separated activity peaks suggested that the neutral beta-N-acetylgalactosaminidase, which was not bound to hydroxyapatite, may be specific to the galactosaminide substrate. The neutral beta-N-acetylglucosaminidase may, on the other hand, have some activity toward the galactosaminide substrate. Both of the neutral enzyme activities were highest during the first postnatal week in rat brain in contrast to the acidic enzyme which showed peak activities during the second and third weeks. These results confirmed and expanded earlier observations by Frohwein and Gatt in calf brain. The relationship of these enzymes to the hexosaminidase C in human tissues is less certain at the present time.     

Characterization of Serine Endopeptidases in Cotyledons of Germinated Vigna mungo Seeds

Vigna mungo seeds. SEP activity was separated into two isoforms by CM-cellulose column chromatography. These forms, termed SEP-1 and SEP-II, showed endopeptidase activities even at acidic pH, suggesting that SEPs are unique serine endopeptidases, since most serine proteases are optimum at neutral pH. Received 14 December 1998/ Accepted in revised form 22 February 1999     

beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.

1. Two forms of beta-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro. 2. beta-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of beta-hexosaminidase during development.     

Identification of factors regulating Escherichia coli 2,3-butanediol production by continuous culture and metabolic flux analysis

2,3-Butanediol (2,3-BDO) is an organic compound with a wide range of industrial applications. Although Escherichia coli is often used for the production of organic compounds, the wild-type E. coli does not contain two essential genes in the 2,3-BDO biosynthesis pathway, and cannot ferment 2,3-BDO. Therefore, a 2,3-BDO biosynthesis mutant strain of Escherichia coli was constructed and cultured. To determine the optimum culture factors for 2,3-BDO production, experiments were conducted under different culture environments ranging from strongly acidic to neutral pH. The extracellular metabolite profiles were obtained using high-performance liquid chromatography (HPLC), and the intracellular metabolite profiles were analyzed by ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry (UPLC/ Q-TOF-MS). Metabolic flux analysis (MFA) was used to integrate these profiles. The metabolite profiles showed that 2,3-BDO production favors an acidic environment (pH 5), whereas cell mass favors a neutral environment. Furthermore, when the pH of the culture fell below 5, both the cell growth and 2,3-BDO production were inhibited.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Senescence in French-bean nodules: Occurrence of different proteolytic activities

A decline in nitrogenase activity (C₂H₂ reduction) of nodules of Phaseolus vulgaris L. cv. Contander was correlated with a decrease in their soluble protein including leghe-moglobin. Concomitantly, two distinct proteolytic activities against leghemoglobin with acidic and alkaline pH optima were detected. The corresponding proteases were purified about 30-fold by ammonium sulfate precipitation, gel filtration and hydroxy-apatite chromatography. Both the acidic (pH optimum 3.5) and the alkaline (pH optimum 8.0) proteases were thiol enzymes. They were characteristic of senescing nodules, whereas only an acidic serine protease was present in functional nodules.     

Increase of intermediate forms of beta-N-acetylhexosaminidase during rat liver development and regeneration.

1. Rat liver beta-N-acetylhexosaminidase was separated into several different molecular forms by DEAE-cellulose chromatography. 2. The subunit composition of the isoenzymes, as well as the similarities to human hexosaminidases, were determined by using the specific active alpha subunit substrate 4-methylumbelliferyl-beta-N-acetylglucosamine-6-sulphate. 3. As in human tissues, the intermediate form lacked the active alpha subunit and resembled hexosaminidase B rather than A. 4. The intermediate form was markedly increased in foetal liver and in regenerating liver after partial hepatectomy. 5. The variations in isoenzyme expression were accompanied by variations in specific activity of hexosaminidase. 6. Sulphated substrate analysis and thermal stability experiments indicated that the rapid cell proliferation had a greater effect on the formation of beta-subunit of hexosaminidase than on that of alpha-subunit.     

Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cells

Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is approximately 680,000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits.     

The nature of the residual alpha-mannosidase in plasma in bovine mannosidosis.

Acidic alpha-mannosidase (EC 3.2.1.24), optimum pH 4.25, is absent from the plasma of Angus calves with mannosidosis, and the residual alpha-mannosidase activity has an optimum pH of 5.5, intermediate between that of the acidic and neutral alpha-mannosidases. This 'intermediate' alpha-mannosidase differs from the acidic form in its kinetic properties, its lack of marked inhibition by EDTA and its thermolability at 55 degrees C and physiological pH. Isoelectric focusing and ion-exchange chromatography show that it exists in at least two forms. The presence of a secondary peak at pH 5.5 in the pH/activity profile of normal plasma and the effect of heating at 55 degrees C indicate that such a form is present in normal plasma. The residual activity in the plasma of a calf with mannosidosis is therefore probably not the product of the defective gene. A differential assay, based on their different stabilities at 55 degrees C, has been developed for measuring the acidic and intermediate alpha-mannosidases in plasma. There was no correlation between the concentrations of the two enzymes in the plasma of Angus cows heterozygous for mannosidosis or in the plasma of normal animals. This precludes the use of the intermediate form as a reference enzyme for the acidic activity in a test for heterozygosity for mannosidosis based on the gene-dosage phenomenon. The concentrations of the intermediate activity were comparable in normal animals and animals homozygous or heterozygous for mannosidosis.     

Purification and characterization of beta-N-acetylhexosaminidase I from human placenta

beta-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A(alpha beta 2) and B[beta beta)2), was purified 1,500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-beta chain-IgG but not to anti-alpha chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4Cer to Gb3Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-beta-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.     

alpha-D-mannosidase forms in chicken liver

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.     

Altered serum alpha-D-mannosidase activity in mucolipidosis II and mucolipidosis III.

Normal human serum contains at least three forms of α-D-mannosidase: an acidic form which has a pH optimum of 4.25, is inhibited by Co²⁺ and is thermostable; an intermediate form, which has a pH optimum of 5.6–5.7, is stimulated by Co²⁺ and is heat labile at 50°C; and a neutral form with a pH optimum of 6.0–6.5. In Mucolipidosis II and III sera, the acidic α-mannosidase activity persists while the intermediate activity is absent or altered. Heating the serum does not affect the pH activity curve, the electrofocusing profile or the response to Co²⁺ of α-mannosidase. During heat inactivation at 55°, 90–100% of the pH 5.6 α-mannosidase activity is lost in normal sera while less than 40% is lost from ML sera. The effect on sera from ML obligate heterozygotes is intermediate. The absent or altered intermediate mannosidase may be responsible for aberrant biochemical properties reported for other glycosidases in Mucolipidosis II and Mucolipidosis III.     

Formation and properties of retinylphosphate galactose.

Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.     

Novel organization and properties of annexin 2-membrane complexes

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.     

Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3-8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.     

Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes.

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.     

Hexosaminidase C in Tay-Sachs and Sandhoff disease.

1. Hexosaminidase C has been purified from human placenta. Complete separation from hexosaminidases A and B was achieved. 2. The following properties of hexosaminidase C differ from those of the A and B isozymes. Presence in the supernatant rather than the lysosomes, neutral pH optimum, higher molecular weight, lack of activity on beta-N-acetylgalactosamine derivatives, and lack of immunological relationship. 3. Hexosaminidase C is active in patients deficient in hexosaminidases A and B and can be recognized by its characteristic electrophoretic mobility. It is concluded that the genetic origin of hexosaminidase C is probably different from that of hexosaminidases A and B.