Cell‐free synthesis of the one‐chain precursor of a major intrinsic protein complex of the small‐intestinal brush border membrane (pro‐sucrase—isomaltase)

Wacker, Hans; Jaussi, Rolf; Sonderegger, P.; Dokow, Monika; Ghersa, Paola; Hauri, Hans‐Peter; Christen, Philipp; Semenza, Giorgio

doi:10.1016/0014-5793(81)80647-3

Cited by 46 publications

(15 citation statements)

References 19 publications

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“…Interestingly, the MI225 000 form of sucrase-isomaltase appeared as a doublet. It is noteworthy that authors in [16], using cell-free translation, obtained two polypeptides of rabbit sucrase-isomaltase of lower A4, than the mature single chain precursor, probably representing non- glycosylated forms of the enzyme. Though the doublet seen here most likely reflects a partial cleavage of the hydrophobic anchor, the possibility also exists that it could represent different but closely related gene products.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of intestinal microvillar proteins

Danielsen

Cowell

1984

FEBS Letters

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The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase‐isomaltase (EC 3.2.1.48–10) and maltase‐glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvillar enzymes was greatly reduced by tunicamycin but could be partially restored by leupeptin, suggesting the existence of a mechanism whereby newly synthesized, malprocessed enzymes are recognized and degraded. In the presence of tunicamycin, polypeptides likely to represent non‐glycosylated forms of the enzymes persisted in the Mg2+‐precipitated membrane fraction, indicating that high mannose glycosylation is essential for transport to the microvillar membrane. Treatment of aminopeptidase N and sucrase‐isomaltase with endo F reduced the size of the high mannose forms approximately to those seen in the presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N‐linked carbohydrate.

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Section: Resultsmentioning

confidence: 99%

Biosynthesis of intestinal microvillar proteins

Danielsen

Cowell

1984

FEBS Letters

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“…Separation of the sucrase and isomaltase polypeptides for sequencing was achieved by preparative polyacrylamide gel electrophoresis on 3 mm thick slab gels using a discontinuous sulphateborate system modified [7] from [ 111. A 20 ~1 aliquot of the sample (20 pg) was 1251-labelled by the chloramine-T procedure [12] and mixed with unlabelled pro-or final sucrase-isomaltase prior to electrophoresis.…”

Section: Separation Of Sucrase and Isomaltase Polypeptidesmentioning

confidence: 99%

“…A singlechain sucrase-isomaltase could also be isolated from the calcium-precipitated membrane fraction, which is expected to contain intracellular and basolateral membranes 161. Finally, the cell-free in vitro translation of single-chain pro-sucrase-isomaltase has been achieved [7,8].…”

Section: Introductionmentioning

confidence: 99%

N‐terminal sequences of pig intestinal sucrase—isomaltase and pro‐sucrase—isomaltase

et al. 1982

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The hog sucrase—isomaltase complex is anchored to the small‐intestinal brush border membrane, as in the rabbit, via a hydrophobic segment located in the N‐terminal region of the isomaltase subunit. The immediate precursor of the ‘final’ sucrase—isomaltase (i.e., pro‐sucrase—isomaltase as prepared from adult hogs whose pancreas had been disconnected from the duodenum) is an amphiphilic single polypeptide chain of M r 260 000–265 000. Its N‐terminal sequence is virtually identical with (not merely homologous to) the corresponding region of the isomaltase subunit of ‘final’ sucrase‐isomaltase. This shows that the isomaltase portion of pro‐sucrase—isomaltase in the N‐terminal ‘half’ of the precursor polypeptide chain. Thus the succession of domains in pro‐sucrase—isomaltase and its mode of anchoring in the membrane could be deduced. On this basis a likely mechanism of biosynthesis and insertion is proposed.

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“…Furthermore, effective methods for isolating microvillar membranes from both intestine and kidney have been described (Schmitz et al, 1973;Booth & Kenny, 1974), as have methods for organ culture of tissue explants for long enough periods to study biosynthetic events (Moscona et al, 1965). Lately, preparations of translatable mRNA from both small intestine (Wacker et al, 1981;Danielsen et al, 1982b) and kidney (Nash & Tate, 1984) have become available, and thus the scientific tools and model systems necessary for a thorough study of the biosynthesis of microvillar proteins are at hand.…”

Section: Introductionmentioning

confidence: 99%

“…A more suitable experimental system for this is cell-free translation of intestinal or kidney mRNA, where the influence of the rough endoplasmic reticulum [in the form of rough microsomes, usually obtained from dog pancreas (Jackson & Blobel, 1977)] can be observed under welldefined conditions. So far, experiments of this type have been performed for rabbit sucrase-isomaltase (Wacker et al, 1981), pig intestinal aminopeptidase N (Danielsen et al, 1983b) and rat y-glutamyl transpeptidase (Nash & Tate, 1984). In these cases, the newly synthesized microvillar enzyme was found predominantly in the membrane fraction of the translation mixture, indicating an interaction between the microsomal membranes and the polypeptide synthesized.…”

Section: Introductionmentioning

confidence: 99%