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Biomedical subjects

J L Stirling

Publications and source records attributed to J L Stirling.

At least 37 records · Page 2Linked to original sources
PubMed1990

Treatment of HL-60 cells with dimethyl sulphoxide inhibits the formation of beta-N-acetylhexosaminidase S.

beta-N-Acetylhexosaminidase of HL-60 cells was separated into two main forms, A and S, by chromatography on DEAE-cellulose. Analysis of developmental changes in the isoenzyme pattern was complicated by the fact that the specific activity of beta-N-acetylhexosaminidase underwent a 6-fold change during the normal growth cycle. Two other lysosomal enzymes, beta-galactosidase and alpha-mannosidase, behaved similarly. Induction of differentiation of HL-60 cells with dimethyl sulphoxide at a low cell density (3 x 10(5) cells/ml) had a greater effect on the abundance of alpha-subunits of beta-N-acetylhexosaminidase, measured with 4-methylumbelliferyl-beta-N-acetylglucosaminide 6-sulphate, than of beta-subunits, measured with 4-methylumbelliferyl-beta-N-acetylglucosamine, and resulted in an isoenzyme profile in which A and B were the major forms, with the levels of form S greatly decreased.

Cell Division↗
PubMed1990

Identification of an intermediate form of beta-N-acetylhexosaminidase in chorionic villi.

A minor form of beta-N-acetylhexosaminidase has been found in chorionic villi, in addition to the major forms A and B. This form does not hydrolyze the 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside-6-sulpha te substrate, is thermostable, has a higher mol mass (120,000) than A and B (100,000) and on analytical isoelectric focusing, it shows a microheterogeneity with values ranging between 6.3 and 7.0. For these characteristics, it resembles beta-N-Acetylhexosaminidase P from pregnancy serum, from which is chromatographically indistinguishable.

Anions↗
PubMed1990

An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.

Blotting, Western↗
PubMed1990

beta-N-acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia.

The spleen from a patient with hairy-cell leukaemia had beta-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an 'extra' form that accounted for 15% of total activity. The 'extra' form hydrolysed the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate, indicating the presence of alpha-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the 'extra' form is entirely composed of alpha-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.

Blotting, Western↗
PubMed1988

Identification of beta-N-acetylhexosaminidase A in mouse tissues with the fluorigenic substrate 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate.

beta-N-Acetylhexosaminidase from mouse tissue was separated into its constituent isoenzymes on DEAE-cellulose and its activity was monitored with 4-methylumbelliferyl-beta-N-acetylglucosamine and 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate. Forms corresponding to the human isoenzymes A (acidic), B (basic) and an 'intermediate' form were present in mouse liver and spleen, whereas in kidney the B and 'intermediate' forms predominated, with A present only as a minor component. In brain the 'intermediate', A and C activities were detected. Testis had predominantly A activity, whereas epididymis, the tissue with the highest specific activity of beta-N-acetylhexosaminidase, had an abundance of the 'intermediate' form, but was almost entirely lacking in the A form.

Animals↗
PubMed1988

Identification of an altered splice site in Ashkenazi Tay-Sachs disease.

Tay-Sachs disease is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (ref. 1). A relatively high frequency of carriers (1/27) of a lethal, infantile form of the disease is found in the Ashkenazi Jewish population, but it is not yet evident whether this has resulted from a founder effect and random genetic drift or from a selective advantage of heterozygotes. We have identified a single-base mutation in a cloned fragment of the HEXA gene from an Ashkenazi Jewish patient. This change, the substitution of a C for G in the first nucleotide of intron 12 is expected to result in defective splicing of the messenger RNA. A test for the mutant allele based on amplification of DNA by the 'polymerase chain rection and cleavage of a DdeI restriction site generated by the mutation revealed that this case and two other cases of the Ashkenazi, infantile form of Tay-Sachs disease are heterozygous for two different mutations. The occurrence of multiple mutant alleles warrants further examination of the selective advantage hypothesis.

Base Sequence↗
PubMed1987

Intermediate forms of human beta-N-acetylhexosaminidase lack activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate.

4-Methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate was purified from a mixture containing its unsulphated precursor. The substrate was used to test for the presence of functional alpha-subunits in 'intermediate' forms of human beta-N-acetylhexosaminidase in samples of normal and pregnancy serum and in extracts of placenta and lymphocytes from a patient with common acute lymphoblastic leukaemia. Intermediate forms in these samples had no activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate, indicating that they lack alpha-subunits.

Chromatography, DEAE-Cellulose↗
PubMed1986

Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

Chromatography, DEAE-Cellulose↗
PubMed1984

Affinity purification and subunit structures of beta-N-acetylhexosaminidases A and B from boar epididymis.

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Animals↗
PubMed1982

Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin.

When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells.

Calcium↗
PubMed1980

The effect of chymotrypsin on the development of Dictyostelium discoideum.

Development of the cellular slime mould Dictyostelium discoideum strain NC4, in the presence of alpha-chymotrypsin (3 mg/ml) is reversibly arrested at the tight aggregate stage (10/12 h). Pronase has a similar effect, but trypsin only retards normal development by about five hours. Normally developing cells are susceptible to alpha-chymotrypsin if they are transferred into its presence at any time up to the tight aggregate stage (10-12 h). Transfer after this stage does not affect the appearance of fruiting body structures in the normal time (24 h). Electron microscopy showed the ultrastructure of alpha-chymotrypsin-blocked aggregates after starvation for 24 h to be consistent with a block at 10-12 h of normal development. Poorly developed prespore vacuoles, having thin incomplete walls and a paucity of electron-dense material, are present in some cells. No angular vacuolated cells characteristic of stalk cells are visible. Fruiting bodies formed in the presence of a alpha-chymotrypsin, either as minority structures when the enzyme is added before 10-12 h of normal development, or as the majority structures on later enzyme addition, were found to be abnormal. Normal stalks were formed but the spores were immature. Prespore vacuoles were present, though disrupted, and the cells were not encapsulated by spore walls. The electronegativity of intact slime mould amoebae was significantly reduced, and material containing L-[6-3H]-fucose and [1-14C]leucine was removed from the cell surface on alpha-chymotrypsin treatment. Few plasma membrane proteins were affected, however, and staining of polyacrylamide gels for glycopeptides using Con A-peroxide binding also showed little change.

Chymotrypsin↗
PubMed1978

Carbohydrate composition of cells and plasma membranes of Dictyostelium discoideum at selected stages of development.

Cells of Distyostelium discoideum representing four developmental stages were auto-analysed for constituent monosaccharides and their compositions compared. Rhamnose, ribose, fucose, glucose, mannose, galactose, glucosamine, galactosamine and an unidentified sugar were recovered after hydrolysis in 2 M HCl for 2 h at 100 degrees C. The relative proportions of the individual sugars were found to vary as a function of development. The largest variations were in the proportions contributed by galactose (from 2% of vegetative cell carbohydrate to 12% of the carbohydrate of fruiting bodies) and galactosamine (present in measurable quantity only in fruiting bodies). Plasma membrane "ghosts" were found to have the same monosaccharide constituents as whole cells, but in different proportions. Mannose contributed over 24% of the total carbohydrate recovered from aggregating cell "ghosts", but only 13% of carbohydrate recovered from "ghosts" prepared from vegetative cells. Galactose was the most abundant sugar recovered from vegetative "ghosts", and was second only to mannose in aggregating "ghosts".

Amino Sugars↗