Solubilization, stabilization and isoelectric focusing of human liver neuraminidase activity

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.

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Degradation of keratan sulphate by β-N-acetylhexosaminidases A and B

Biochemical Journal ◽

10.1042/bj1930811 ◽

1981 ◽

Vol 193 (3) ◽

pp. 811-818 ◽

Cited By ~ 26

Author(s):

T Ludolph ◽

E Paschke ◽

J Glössl ◽

H Kresse

Keyword(s):

Isoelectric Focusing ◽

Human Liver ◽

Thermal Inactivation ◽

Sandhoff Disease ◽

Chromogenic Substrate ◽

Polymeric Substrate ◽

Keratan Sulphate ◽

Human Enzyme ◽

Tay Sachs Disease

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay–Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH–activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.

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Purification and comparison of the structures of human liver acidic α-d-mannosidases A and B

Biochemical Journal ◽

10.1042/bj2330065 ◽

1986 ◽

Vol 233 (1) ◽

pp. 65-72 ◽

Cited By ~ 31

Author(s):

S H Cheng ◽

S Malcolm ◽

S Pemble ◽

B Winchester

Keyword(s):

Molecular Mass ◽

Isoelectric Focusing ◽

Concanavalin A ◽

Human Liver ◽

Large Subunit ◽

Small Subunit ◽

Single Locus ◽

Structure And Properties ◽

Molecular Masses ◽

High Mannose

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.

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Isoelectric focusing of human liver alkaline phosphatase

Biochemical Journal ◽

10.1042/bj1180299 ◽

1970 ◽

Vol 118 (2) ◽

pp. 299-302 ◽

Cited By ~ 28

Author(s):

A. L. Latner ◽

Mary E. Parsons ◽

A. W. Skillen

Keyword(s):

Alkaline Phosphatase ◽

Density Gradient ◽

Isoelectric Focusing ◽

Isoelectric Point ◽

Human Liver ◽

Sucrose Density Gradient ◽

Ph Gradient ◽

Alkaline Phosphatases ◽

Carrier Ampholytes

1. Isoelectric focusing of human liver alkaline phosphatase in a sucrose density gradient with LKB Ampholine as carrier ampholytes is described. 2. Problems due to the chelating properties of the ampholytes and the pH gradient were examined. 3. A reactivation procedure to counter these effects was devised that can probably be used for other alkaline phosphatases. 4. The isoelectric point of human liver alkaline phosphatase was found to be pH3.9.

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Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

Biochemical Journal ◽

10.1042/bj2290679 ◽

1985 ◽

Vol 229 (3) ◽

pp. 679-685 ◽

Cited By ~ 3

Author(s):

R L Hopfer ◽

J A Alhadeff

Keyword(s):

Human Brain ◽

Isoelectric Focusing ◽

Human Liver ◽

Gel Filtration ◽

Compelling Evidence ◽

Supernatant Fluid ◽

Ph Optimum ◽

Triton X ◽

Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

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Antibody-affinity purification of novel α-l-fucosidase from mouse liver

Biochemical Journal ◽

10.1042/bj2450589 ◽

1987 ◽

Vol 245 (2) ◽

pp. 589-593 ◽

Cited By ~ 10

Author(s):

L D Laury-Kleintop ◽

I Damjanov ◽

J A Alhadeff

Keyword(s):

Isoelectric Focusing ◽

Gel Electrophoresis ◽

Human Liver ◽

Mouse Liver ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Antibody Affinity ◽

Native Polyacrylamide Gel Electrophoresis ◽

Sepharose 4B

Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.

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Glutathione transferase isoenzymes from human prostate

Biochemical Journal ◽

10.1042/bj2710481 ◽

1990 ◽

Vol 271 (2) ◽

pp. 481-485 ◽

Cited By ~ 26

Author(s):

C Di Ilio ◽

A Aceto ◽

T Bucciarelli ◽

S Angelucci ◽

M Felaco ◽

...

Keyword(s):

Isoelectric Focusing ◽

Immunohistochemical Staining ◽

Human Liver ◽

Glutathione Transferase ◽

Human Prostate ◽

Immunoblotting Analysis ◽

Qualitative And Quantitative ◽

Quantitative Expression ◽

Close Relationship ◽

Liver And Kidney

By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.

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Isoelectric focusing of isoenzymes of human liver α-l-fucosidase

FEBS Letters ◽

10.1016/0014-5793(75)81076-3 ◽

1975 ◽

Vol 54 (1) ◽

pp. 89-92 ◽

Cited By ~ 40

Author(s):

Robin Thorpe ◽

Donald Robinson

Keyword(s):

Isoelectric Focusing ◽

Human Liver

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Properties of human tissue isoferritins

Biochemical Journal ◽

10.1042/bj1730969 ◽

1978 ◽

Vol 173 (3) ◽

pp. 969-977 ◽

Cited By ~ 92

Author(s):

M Wagstaff ◽

M Worwood ◽

A Jacobs

Keyword(s):

Ion Exchange ◽

Isoelectric Focusing ◽

Iron Content ◽

Iron Uptake ◽

Human Liver ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Normal Tissues ◽

Multiple Blood ◽

Liver Ferritin

1. Human liver ferritin was separated by preparative isoelectric focusing into six fractions. 2. Except for the least acidic fraction the reactivity with antibody against spleen ferritin increased with rising pI, but with antibody against heart ferritin the reactivity decreased. 3. The highest iron content was found in the most acidic isoferritins and progressively decreased with rising pI. 4. Iron uptake was studied in apoferritin prepared from heart and liver ferritin fractions separated by ion-exchange chromatography. There was good correlation between the rate of iron uptake and pI. The most acidic fractions took up iron more rapidly than did the more basic ones. 5. Ferritin was prepared from heart, liver, spleen and kidney. There was little difference on isoelectric focusing between ferritin obtained from normal tissues and the corresponding iron-loaded tissues from patients who had received multiple blood transfusions. The iron-loaked heart ferritin invariably contained relatively more of the basic isoferritins. Normal and iron-overloaded heart ferritins were separated into isoferritin fractions by ion-exchange chromatography, and in each case there was a fall in iron content as the pI increased. The iron content of ferritin from the iron-overloaded heart was higher throughout than that from normal heart. 6. There is a relationship between the rate of iron uptake by apoferritin and pI, and this probably accounts for the variation in iron content of the isoferritins found in human liver and heart.

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Multiple Molecular Forms of Human Heart Cytoplasmic Aspartate Aminotransferase

Clinical Science ◽

10.1042/cs0620337 ◽

1982 ◽

Vol 62 (3) ◽

pp. 337-339 ◽

Cited By ~ 6

Author(s):

F. Y. Leung ◽

A. R. Henderson

Keyword(s):

Isoelectric Focusing ◽

Aspartate Aminotransferase ◽

Human Liver ◽

Human Heart ◽

Specific Activity ◽

Molecular Forms ◽

Aminotransferase Activity ◽

Multiple Molecular Forms ◽

Isoelectric Points

1. Cytoplasmic aspartate aminotransferase was isolated and purified from human heart with a final specific activity of 236 units/mg of protein. 2. Three distinct peaks of aspartate aminotransferase activity were detected by isoelectric focusing with isoelectric points of 5.46, 5.60 and 5.71. Two minor subforms were also noted as shoulder patterns with pI 5.2 and 5.8. 3. These electrophoretic characteristics are similar to previous findings of multiple molecular forms detected in human liver and erythrocytes.

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