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An improved method for diagnosis of gaucher’s disease using skin fibroblasts and a chromogenic substrate for glucocerebrosidase
ABSTRACTS OF A N N U A L MEETIN<;
1980
167
sulphatc. All of these disaccharides contain an x-glucosaminide-N-sulphateresidue linked to either idosc (GlcNSIdo). idosc-2-sulphate (GlcNS-Ido(OS)), gluconic acid (GlcNS-GlcOA) or 2.5 anhydroidonic acid (GlcNSanldOA). Normal fibroblasts de-N-sulphated all but the lirst at approximately I 4 pmol min nip protcin, but Sanfihppo A fibroblasts were inactive ( < 1 fmol~ininjmg). Similarly, the corresponding N-acetylated disaccharides GlcNAc-Ido and GlcNAc-GlcOA wci-c dc-N-acctylatcd by normal fibroblast homogenates (3 pinol/min/mg and 17 pmol/mintmg respectively) but were not by Sanfilippo B fibroblasts. In the presence of Acetyl-CoA, GlcN-ldo aiid GlcN-GlcOA were nctively acctylatcd by thc N acetyltninsferase activity of normal fibroblasts at 10 pinoljmininig: this enzyme is deficient i n Snnfilippo typc C. We conclude these disaccharidcs are convenient iind significant improveinents upon the convcntioiiiil dingnostic substrates for the assay of the Sanfilippo enzymes. AN IMPROVED METHOD FOR DIAGNOSIS OF GAUCHER’S DISEASE USING SKIN FIBROBLASTS AND A CHROMOGENIC SUBSTRATE FOR GLUCOCEREBROSIDASE
R. .I.BARNS & A. E. CLAGIJFP a / k o k ~Dc,pc/r/n?cw7r. ~~ Roj,r// Hri.t/Jr/nc’Ifo.spittr/ The glucosylcei-ainideanalogue, 2-N-hcxadecanoylamino-4-nitrophenyl-/l-D-glticopyr~1n~~sidc ( H N G l u ) has bccii shown to be B suitable glucocerebrosidase subhti-ate for the diagnosis of Gauchcr’s disease. We report ;I procedure giving u p to 20-fold more activity in normal skin fibroblasts than the reportcd procedure. but still ~howingncgligiblc activity in Gauclier’s disease fibroblasts. The assay involves the incubation ofthe fibroblast extract with HNGlu (1.08 m M ) a t pH 5.0 in the presence o f sodium taurocholate (9 3 mM) and oleic acid (1.06 mM) as activators. Normal fibroblasts ~ h o w c d; i n activity 01’ 3.0-9.3 Ulg protein, with a K m (apparent) of0.15 m M . Fibroblasts from a patient with infantile Gauchcr’s discac showed minimal activity from pH 4.0 6.4. indicating the absence of any other acid /I glucosidaac activity tow:ird\ HNGlti. At pH 5.0. the patient’s activity was 0.3 Uig protciii, while his parcnts had activities o l 3 . S U,g protein and I .5 U.gprotein. This reduced activity resulted fi-om H marked decrease in V inax with no change iii Kin (;ippareiit) The assay appears suilable for the diagnosis o l Gaucher’s disease a n d , possibly. for Gauchci-‘s disease hcterozygotcs. ISOLATION AND USE OF A NEW NATURAL SUBSTRATE FOR THE DIAGNOSIS OF MPS VII
VlVlENNE MULLER & JOHN H o ~ w o o u Dc~/~arrnicnrof C/7cv77ic,c~/Prrf/7o/o,q,1~, T/w A r / c / r r i r / c , C’hildwn’,~ lIospitu/ Mucopolysaccharidosis Type V [ I is characterized by a sevcre deficiency of the en7yine /~-D-gI~icuroiiitlasc. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range olphcnotypcs. from cxlreiiicly mild to severe. Diagnosis ofthe enzyme deficiency has usually been made using artificial substrates such as 4-mcthyluinhclliferyl/I-D-glucuronide (MuGlcUA), and p-nitrophenyl-g~D-glucuronide. We now report the isolation o f a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-aulphatc (GMs) derived from the controlled degradation of heparin. Under optimal a s s a y conditions for norinal h k i n fibroblast homogenates, this natural substrate gives /i-D-glucuronidase activities of 0-2.4. 9 I7 aiid 38 56 pmol:min/nig protein lor homozygote affected, heterozygotc and normal ccll lines, respectively. Compared with the artificial substrate MLIGI~UA, normal activity measured with GMb demonstfiitcs ;I similar pH profile and time course, but a changed response to protein levels, greater heat scnsitivity, and also Iowci- K,,, and V,,,,,, values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs). a natural subsirarc of r-Liduronidase. Howcver, in contrast to a-L-iduronidase, [&D-glucuronidase is inhibited only slightly by SO: or Cu’ ’ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that [I-D-glucuronid lack the specific SO: and Cu2+ binding sites postulated for a-L-iduronidase. u,-ANTITRYPSIN (PI) SUBTYPING
JOHN MU LLEY
C.vtogenefic.s C/ni/, Dc./mr/mcw/ of H / , s t o / ~ r / / / t o / 7%c, o ~ ~ ~.4t/c,/tr/r/r’ . (‘/ri/(/rc,,7:v
MOS/)itU/
a , A T is the major regulator ofprotease activity in humans. Genetic heterogeneity is determined a t the PI locus with at least 32 codominant alleles. Many variants are associated with reduced a , A T serum levels aiid mo\i arc infrequent, although PI*S, P/*Z. PI*F and PI*] are relatively common. The distribution of PI M subtypes from Adelaide blood donors was determined by a high resolution isoelcctric focusing technique. Allele frequencies were: P P M I = 0.65. P P M 2 = 0.14. P I * M 3 = 0.13. P I * S = 0.061,
1980
167
sulphatc. All of these disaccharides contain an x-glucosaminide-N-sulphateresidue linked to either idosc (GlcNSIdo). idosc-2-sulphate (GlcNS-Ido(OS)), gluconic acid (GlcNS-GlcOA) or 2.5 anhydroidonic acid (GlcNSanldOA). Normal fibroblasts de-N-sulphated all but the lirst at approximately I 4 pmol min nip protcin, but Sanfihppo A fibroblasts were inactive ( < 1 fmol~ininjmg). Similarly, the corresponding N-acetylated disaccharides GlcNAc-Ido and GlcNAc-GlcOA wci-c dc-N-acctylatcd by normal fibroblast homogenates (3 pinol/min/mg and 17 pmol/mintmg respectively) but were not by Sanfilippo B fibroblasts. In the presence of Acetyl-CoA, GlcN-ldo aiid GlcN-GlcOA were nctively acctylatcd by thc N acetyltninsferase activity of normal fibroblasts at 10 pinoljmininig: this enzyme is deficient i n Snnfilippo typc C. We conclude these disaccharidcs are convenient iind significant improveinents upon the convcntioiiiil dingnostic substrates for the assay of the Sanfilippo enzymes. AN IMPROVED METHOD FOR DIAGNOSIS OF GAUCHER’S DISEASE USING SKIN FIBROBLASTS AND A CHROMOGENIC SUBSTRATE FOR GLUCOCEREBROSIDASE
R. .I.BARNS & A. E. CLAGIJFP a / k o k ~Dc,pc/r/n?cw7r. ~~ Roj,r// Hri.t/Jr/nc’Ifo.spittr/ The glucosylcei-ainideanalogue, 2-N-hcxadecanoylamino-4-nitrophenyl-/l-D-glticopyr~1n~~sidc ( H N G l u ) has bccii shown to be B suitable glucocerebrosidase subhti-ate for the diagnosis of Gauchcr’s disease. We report ;I procedure giving u p to 20-fold more activity in normal skin fibroblasts than the reportcd procedure. but still ~howingncgligiblc activity in Gauclier’s disease fibroblasts. The assay involves the incubation ofthe fibroblast extract with HNGlu (1.08 m M ) a t pH 5.0 in the presence o f sodium taurocholate (9 3 mM) and oleic acid (1.06 mM) as activators. Normal fibroblasts ~ h o w c d; i n activity 01’ 3.0-9.3 Ulg protein, with a K m (apparent) of0.15 m M . Fibroblasts from a patient with infantile Gauchcr’s discac showed minimal activity from pH 4.0 6.4. indicating the absence of any other acid /I glucosidaac activity tow:ird\ HNGlti. At pH 5.0. the patient’s activity was 0.3 Uig protciii, while his parcnts had activities o l 3 . S U,g protein and I .5 U.gprotein. This reduced activity resulted fi-om H marked decrease in V inax with no change iii Kin (;ippareiit) The assay appears suilable for the diagnosis o l Gaucher’s disease a n d , possibly. for Gauchci-‘s disease hcterozygotcs. ISOLATION AND USE OF A NEW NATURAL SUBSTRATE FOR THE DIAGNOSIS OF MPS VII
VlVlENNE MULLER & JOHN H o ~ w o o u Dc~/~arrnicnrof C/7cv77ic,c~/Prrf/7o/o,q,1~, T/w A r / c / r r i r / c , C’hildwn’,~ lIospitu/ Mucopolysaccharidosis Type V [ I is characterized by a sevcre deficiency of the en7yine /~-D-gI~icuroiiitlasc. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range olphcnotypcs. from cxlreiiicly mild to severe. Diagnosis ofthe enzyme deficiency has usually been made using artificial substrates such as 4-mcthyluinhclliferyl/I-D-glucuronide (MuGlcUA), and p-nitrophenyl-g~D-glucuronide. We now report the isolation o f a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-aulphatc (GMs) derived from the controlled degradation of heparin. Under optimal a s s a y conditions for norinal h k i n fibroblast homogenates, this natural substrate gives /i-D-glucuronidase activities of 0-2.4. 9 I7 aiid 38 56 pmol:min/nig protein lor homozygote affected, heterozygotc and normal ccll lines, respectively. Compared with the artificial substrate MLIGI~UA, normal activity measured with GMb demonstfiitcs ;I similar pH profile and time course, but a changed response to protein levels, greater heat scnsitivity, and also Iowci- K,,, and V,,,,,, values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs). a natural subsirarc of r-Liduronidase. Howcver, in contrast to a-L-iduronidase, [&D-glucuronidase is inhibited only slightly by SO: or Cu’ ’ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that [I-D-glucuronid lack the specific SO: and Cu2+ binding sites postulated for a-L-iduronidase. u,-ANTITRYPSIN (PI) SUBTYPING
JOHN MU LLEY
C.vtogenefic.s C/ni/, Dc./mr/mcw/ of H / , s t o / ~ r / / / t o / 7%c, o ~ ~ ~.4t/c,/tr/r/r’ . (‘/ri/(/rc,,7:v
MOS/)itU/
a , A T is the major regulator ofprotease activity in humans. Genetic heterogeneity is determined a t the PI locus with at least 32 codominant alleles. Many variants are associated with reduced a , A T serum levels aiid mo\i arc infrequent, although PI*S, P/*Z. PI*F and PI*] are relatively common. The distribution of PI M subtypes from Adelaide blood donors was determined by a high resolution isoelcctric focusing technique. Allele frequencies were: P P M I = 0.65. P P M 2 = 0.14. P I * M 3 = 0.13. P I * S = 0.061,