Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver

1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.     

Studies of the heterogeneity of carp liver acid phosphatases: acid phosphatase--I. Subunit structure and carbohydrate composition

The three molecular forms of the carp liver acid phosphatase (AcPase) were shown to be dimeric proteins, two of them differing in molecular weights. An activating effect of ConA binding on the AcPases has been observed. AcPase I and AcPase II showed a mol. wt of 122,500 and of 58,884 +/- 3000 for their subunits. It is assumed that AcPase I is a sialylated derivative of AcPase II. AcPase III has a mol. wt of 93,132 and the two subunits of 46,556 +/- 4000. A homogeneous AcPase I was obtained and its carbohydrate composition is presented.     

Acid phosphatase from liver of the frog Rana esculenta, separation and partial characterization of multiple forms.

1. Acid phosphatase (AcPase) from liver of the frog, Rana esculenta has been isolated and purified. The enzyme is heterogeneous, showing 4 activity zones on disc electrophoresis. The AcPase was separated into 3 peaks on DEAE-cellulose. Peak A corresponding to the electrophoretic AcPase IV represents an extensively purified enzyme form. 2. The separated enzyme forms are change isomers with a molecular weight of about 33,000. They differ markedly in substrate requirements and sensitivity towards activators and inhibitors. All of them are highly activated by dithiothreitol, show a rather restricted substrate specificity, and marked activity against ATP.     

Isolation and characterization of a homogeneous acid phosphatase from catfish liver

A homogeneous, tartrate-inhibitable acid phosphatase (AcPase) was obtained from the liver of channel catfish (Ictalurus punctatus) by the use of Affi Gel-10-coupled aminohexyltartramic acid affinity chromatography. The enzyme has a molecular weight of 82,500 and is a dimer consisting of two apparently equivalent subunits with subunit weights of 35,000 +/- 3000. Amino acid composition data are presented and compared with those of mammalian acid phosphatases. Data suggest that the enzyme is a metalloacid phosphatase. Catfish liver AcPase exhibits two molecular forms with pI 5.66 and 5.37 which were separated by chromatofocusing. A spontaneous conversion of the less acidic form to a more acidic form was observed and this conversion was accompanied by a decreased sensitivity towards tartrate inhibition.     

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.     

Amino acid composition and immunochemical properties of AcPase III and AcPase IV representing glycoforms of the lower molecular weight, tartrate-resistant acid phosphatase of the frog liver.

1. Amino acid composition and immunological properties of the frog liver LMW AcPase forms: AcPase III and IV were examined. 2. AcPase III and IV show nearly identical amino acid composition and close immunological similarity. 3. These results indicate protein identity of both the enzyme forms and together with our previous data [Kubicz A., Szalewicz A. and Chrambach A., Int. J. Biochem. 23, 413-419 (1991)] demonstrate that generation of AcPase III and IV is a modification of the same enzyme protein by glycosylation processes. 4. Differences in immunoreactivity between AcPase III and IV were observed and discussed to be due to their altered conformations.     

Deglycosylation by trifluoromethanesulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine.

Endopeptidase-24.11, an integral microvillar membrane enzyme, exists in differently glycosylated forms when purified from pig kidney and intestine [Fulcher, Chaplin & Kenny (1983) Biochem. J. 215, 317-323]. When these glycoproteins, and another form of the kidney enzyme prepared from the Yucatan dwarf strain of piglet, were treated, under controlled conditions, with trifluoromethanesulphonic acid, the proteins were freed of carbohydrate and all had the same apparent subunit Mr (77 000) even though the untreated forms varied from Mr 89 000 to Mr 95 000.     

Spontaneous conversion of carp liver AcPase I and some properties of its asialo-form

Evidence is given for a spontaneous conversion of AcPase I to a more basic form due to autolytic loss of sialic acid. To investigate whether the presence of sialic acid in the enzyme molecule is of any physiological significance some physico-chemical properties of an enzymatically obtained asialo-AcPase I were studied. It has been shown that the asialo-form retained the properties of the native enzyme in respect to thermostability and pH-stability but modified slightly the hydrolytic activities of some phosphorylated compounds, and this might be of significance under physiological conditions.     

The role of N-glycosylation for the plasma clearance of rat liver secretory glycoproteins

The clearance of total rat liver secretory glycoproteins and of alpha 1-acid glycoprotein carrying no or different types of oligosaccharide side chains was studied in vivo and in the isolated perfused rat liver. In order to obtain unglycosylated or differently glycosylated forms of secreted glycoproteins, rat hepatocyte primary cultures were incubated with various inhibitors of N-glycosylation. Tunicamycin was used for the synthesis of unglycosylated (glyco)proteins, the mannosidase I inhibitor 1-deoxymannojirimycin for the synthesis of high-mannose type and the mannosidase II inhibitor swainsonine for the synthesis of hybrid-type glycoproteins. Glycoproteins carrying carbohydrate side chains of the complex type were synthesized by control hepatocytes. In vivo and in the perfused rat liver, high-mannose-type glycoproteins were cleared at the highest rate, followed by unglycosylated and hybrid-type glycoproteins. The lowest clearance rate was found for the glycoproteins with carbohydrate side chains of the complex type. For the highly glycosylated alpha 1-acid glycoprotein the differences in clearance rates were more pronounced. The following plasma half-lives were determined in vivo: complex type, 100 min; hybrid type, 15 min; unglycosylated form, 5 min; and high-mannose type less than 1 min. In the recirculating perfused liver 28% of complex-type alpha 1-acid glycoprotein, 40% of hybrid type, 47% of unglycosylated and 93% of high-mannose-type alpha 1-acid glycoprotein were removed from the perfusate within 2 h. It is concluded that N-glycosylation and processing to complex-type oligosaccharides seems to be of great importance for the circulatory life time of plasma glycoproteins.     

Role of carbohydrate content on the properties of galactose oxidase from Dactylium dendroides

The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.     

Immunopurification and characterization of human alpha-L-iduronidase with the use of monoclonal antibodies.

alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.     

Polymorphism of rat gastric mucosal histidine decarboxylase: effects of the coenzyme and sulfhydryl groups

Several factors are examined for their implication in the charge heterogeneity and form conversion of rat gastric mucosal histidine decarboxylase. The apoenzyme and the holoenzyme are undistinguishable with respect to their pI and to the distribution of enzyme activity in the three forms. The latter are not produced by differential coenzyme binding. Studies for glycoprotein characterization provide evidence that the heterogeneity does not arise from enzyme-bound carbohydrate. Oxidative or reductive environments change the distribution between forms without modifying the molecular weight. Conversion of form III to forms I and II can be effected by treatment with dithiothreitol. A similar loss of negatively charged form occurs upon ageing and is not prevented by an alkylating agent. All three forms show equal sensitivity to N-ethylmaleimide and dithiothreitol inhibitions. The oxidation-reduction state of exposed sulfhydryl groups may be responsible at least in part for the charge heterogeneity.     

The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.     

Evidence for two activated forms of pyruvate kinase from Bacillus stearothermophilus in the presence of ribose 5-phosphate

Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined. Two activated forms of this enzyme could be distinguished, depending on the R5P concentration. One form (Form I) was observed at about 10(-5) M R5P. It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP). The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP. The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II. In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM. The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it. The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I. With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased. The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II. These results indicate that the two activated forms distinguished kinetically differ in their conformations, too. The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.     

Effect of the co-existence of galactosyl and phosphomannosyl residues on β-hexosaminidase on the processing and transport of the enzyme in mucolipidosis I fibroblasts

Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compare the properties of intra- and extracellular β-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete β-hexosaminidase when maintained in serum-free medium. Using the specifity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted β-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that β-hexosaminidase molecules contained phosphomanosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of β-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.     

Acid phosphatases from liver of Rana esculenta. Subcellular localization and partial characterization of multiple forms

1. Four acid phosphatases (AcPase I, II, III and IV) were found in the liver of the frog Rana esculenta. 2. AcPases I, II, III, and IV were associated with the microsomal, mitochondrial-lysosomal, nuclear and soluble fractions respectively and showed apparent molecular weights of about 240,000, 110,000, 38,000 and 17,000. 3. All the enzymes show acid pH optima, and similar Km values for p-nitrophenylphosphate. 4. AcPases I, II, and III hydrolyze most of the common phosphate esters whereas AcPase IV hydrolyzes effectively only p-nitrophenylphosphate, phenylphosphate and flavine mononucleotide. 5. AcPases I and II are inhibited by NaF and tartrate. AcPases III and IV are tartrate resistant. 6. Temperature inhibits AcPases I, II, IV, whereas it activates AcPase III.