Immunoelectrophoretic Studies on Human Small Intestinal Brush Border Proteins. The Residual Isomaltase in Sucrose Intolerant Patients

Skovbjerg, Hanne; Krasilnikoff, P. A.

doi:10.1203/00006450-198103000-00004

Cited by 20 publications

(16 citation statements)

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“…150-200 p,1 of the supernatants containing approximately 300 mD sucrase and 4 mg mucosal protein was run in line immunoelectrophoresis (10) against specific anti-human-SI-IgG (300 p,g/ml gel) as described in (14). The fetal SI crossreacted immunologically with adult SI as it precipitated with the IgG made by immunizing rabbits with purified adult SI.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…After washing and pressing of the immunoelectrophoretic plate the immunoprecipitates (visible without staining) were excised. Rocket immunoelectrophoresis (16) of the supernatants against the specific anti-human-SI-IgG was performed as in (14).Polyacrylamide gel electrophoresis. Gel electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulphate was performed by the method of Laemmli (II).…”

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confidence: 99%

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High Molecular Weight Pro-Sucrase-Isomaltase in Human Fetal Intestine

Skovbjerg

1982

Pediatr Res

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SummarySucrase-isomaltase (SI) was isolated by immunoprecipitation from the intestines of human fetuses of [16][17][18][19][20] wk of gestational age as one large polypeptide (pro-SI) with a molecular weight of 260,000. Treatment with elastase converted the pro-SI into two polypeptides of apparent molecular weights as sucrase and isomaltase from normal adult intestine (M. 140,000 and 150,000, respectively). The pro-SI had full enzymatic activity. SpeculationThe results indicate that human sucrase-isomaltase is synthesized as a single chain and that the pancreatic proteases play a significant role in the final processing of the molecule. The pro-SI is proposed to be present also in patients suffering from pancreatic insufficiency.Sucrase-isomaltase (SI) is a digestive enzyme responsible for all the sucrase activity, nearly all the isomaltase activity, and for most of the maltase activity (-70%) of the small intestine. SI is localized with its major part exposed to the luminal content of the intestine and anchored to the small intestinal brush border membrane by a small hydrophobic segment [for review see (12)]. In the majority of mammals including man, SI occurs as an enzyme complex built up of two polypeptides one of which has sucrase the other isomaltase activity.By observation on pigs whose pancreas has been totally disconnected it has been shown that SI is synthesized as a single-chain, assigned pro-SI (13). Treatment of this pro-SI with pancreatic proteases splits it into two polypeptides of apparent molecular sizes similar to those of the "final" SI (13), suggesting that the final step in the processing of SI occurs at the luminal surface of the intestine by action of the pancreatic enzymes.In the developing human fetus zymogen granules have been detected in the pancreas as early as the 5th month of gestation [for review see (9)]; however, the intestinal enterokinase, which is the activator for trypsinogen, does not appear until the 26-30 wk of gestation (I). This implies that the fetal pancreas does not have any proteolytic activity until this time. Fetal intestines before 26 wk of gestational age therefore provide a unique model to investigate if the biosynthesis of the human SI is likely to proceed via a large pro-SI and the possible role of pancreatic proteases in the processing of the SI. MATERIALS AND METHODSAnti-human-SI-IgG was obtained against normal, adult SI as previously described (14).Two human fetuses of 16-20 wk of gestational age were obtained after abortus provocatus. Their full length intestines were immediately cut out and frozen at minus 80°C until use. As control material small jejunum pieces of normal adult intestine were used (15).Solubilization of the brush border proteins. The proximal part of each fetal intestine (approximately 0.3 g) was homogenized in crushed ice in 4 volumes of a 50 mM potassium phosphate buffer, pH 6.0, containing 1% Triton X-100 and aprotinin 28 p,g X ml-The homogenate was incubated at 4°C for I h and subsequently centrifuged 50,000 X g for I h at 4°C. The sup...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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High Molecular Weight Pro-Sucrase-Isomaltase in Human Fetal Intestine

Skovbjerg

1982

Pediatr Res

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“…Recently, single amino acid substitution was identified in sucrase subunit in a patient with CSID (34). On the other hand, human cases with isolated sucrase deficiency are also reported, however, the precise molecular basis of this abnormality has not been elucidated (35,36). The present report is the first to investigate the molecular basis of isolated sucrase deficiency in the mammalian species.…”

Section: Discussionmentioning

confidence: 86%

Molecular Cloning of Sucrase-Isomaltase cDNA in the House Musk Shrew Suncus murinusand Identification of a Mutation Responsible for Isolated Sucrase Deficiency

Ito

Hayashi

Ohmori

et al. 1998

Journal of Biological Chemistry

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Isolated sucrase deficiency has been demonstrated in a line of house musk shrew, Suncus murinus (laboratory name: suncus). This animal belongs to the order Insectivore and is phylogenetically different from ordinarily used laboratory animals. They are believed to have evolved with mainly animal food without sucrose. To study the molecular basis of the sucrase deficiency in suncus, we cloned 6.0-kilobase (kb) sucrase-isomaltase (SI, EC 3.2.1.48 -10) cDNA from suncus intestinal cDNA library. The cDNA clone contained a 5442-base pair (bp)-long open reading frame preceded by an in frame termination codon. The deduced 1813-amino acid sequence showed 68.6, 71.2, and 74.7% similarity with those of rat, rabbit, and human, respectively. A cleavage site between isomaltase and sucrase as well as the region surrounding the catalytic sites for sucrase and isomaltase were conserved among the species. Out of 18 potential N-linked glycosylation sites, 5 were common among all 4 species. In the connecting segment which was enriched with O-linked glycosylation sites in the other species, only two sites were present in suncus. Northern blot analysis revealed that the 6.0-kb SI mRNA was expressed in the KAT line with intact sucrase-isomaltase activity. In contrast, 3.0-kb SI mRNA was expressed in suncus of the MI line with isolated sucrase deficiency. The 3.0-kb mRNA cosegregated with sucrase deficiency phenotype as an autosomal recessive trait. Sequence analysis revealed a 2-nucleotide deletion at position 2767-2768, which results in a frameshift and an immature termination codon. The cDNA of the MI line diverged from that of the KAT line at position 2865, having an 18-bp unique sequence followed by a poly(A) tail. The mutant cDNA encodes 922 amino acid residues which preserves the region for isomaltase but lacks that for whole sucrase. While the cells transfected with the plasmids expressing SI in the KAT line showed both sucrase and isomaltase activity, the plasmids expressing MI line cDNA showed only isomaltase activity. Thus it was concluded that the mutation in the SI gene was responsible for isolated sucrase deficiency in the MI line.Mammals of the order Insectivore have received attention as possible laboratory animals which are phylogenetically different from ordinarily used laboratory animals, such as the mouse, rat, hamster, and rabbit (1, 2). Both primates and rodents apparently originated from Insectivore and then evolved separately (3). In 1976, Oda and Kondo succeeded in rearing and breeding the house musk shrew, Suncus murinus, mammal belonging to the family Soricidae of Insectivore (4). Thereafter, several laboratory lines of suncus which originated from different wild populations in Asian regions have been developed in Nagoya, Japan, and a number of studies have been carried out on the morphology, endocrinology, and physiology of this animal (1). As a result, several lines of suncus were established as a model of sucrase deficiency. It was further established that hereditary sucrase deficiency in suncus was transmi...

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“…Table 1 identifies the proposed clinical phenotypes based on the reduction in small intestinal disaccharidase activities and dietary tolerance among those patients with CSID followed by the support group. The range of mucosal biopsy activities is taken from Table 2, which summarizes 3 patterns of CSID disaccharidase mucosal enzyme deficiencies described in the literature (1)(2)(3)(4)(5)(6)(7) and in this workshop (8)(9)(10)(11) and makes a tentative correlation with the dietary tolerances in Table 1. One goal of future research is to confirm whether these 5 dietary phenotypes correlate with 3 mutant genotypes of SI.…”

Section: Proposed Phenotypes Based On Intestinal Disaccharidase Activ...mentioning

confidence: 99%

Phenotypic Observations by the CSID Dietary and Medical Support Group

Slawson¹

2012

J. pediatr. gastroenterol. nutr.

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they may also offer relief for transient deficiencies such as may occur with small bowel injury.The strengths of these data lie in the large sample size that is nationally representative and the consistent and experienced personnel who performed the analyses; the enzyme assay has been in use and has not changed in decades. The weaknesses of these data lie in the lack of clinical correlation, the selection by the endoscopists of a young age group from which samples were obtained, the inability to control for sample integrity throughout the entire process of obtaining the samples, and handling and shipping. The analyses were performed manually, and human error is always a possibility. We conclude that the most common disaccharidase deficiency is lactase followed by pandisaccharidase. Sucrase deficiency was rare in these samples.

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Immunoelectrophoretic Studies on Human Small Intestinal Brush Border Proteins. The Residual Isomaltase in Sucrose Intolerant Patients

Cited by 20 publications

References 8 publications

High Molecular Weight Pro-Sucrase-Isomaltase in Human Fetal Intestine

High Molecular Weight Pro-Sucrase-Isomaltase in Human Fetal Intestine

Molecular Cloning of Sucrase-Isomaltase cDNA in the House Musk Shrew Suncus murinusand Identification of a Mutation Responsible for Isolated Sucrase Deficiency

Phenotypic Observations by the CSID Dietary and Medical Support Group

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