Subunits of the Small‐Intestinal Sucrase · Isomaltase Complex and Separation of Its Enzymatically Active Isomaltase Moiety

Cogoli, Augusto; Eberlé, Alex N.; Sigrist, Hans; Joss, Christine; Robinson, E A; Mosimann, Hugo; Semenza, Giorgio

doi:10.1111/j.1432-1033.1973.tb02652.x

Cited by 82 publications

(45 citation statements)

References 24 publications

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“…We also present additional evidence that in the mild alkaline treatment which leads to the preparation of isomaltase (as reported previously [4,6]) no action of contaminating proteases is involved.…”

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confidence: 50%

“…Detergent-free polyacrylamide-gel electrophoresis was performed as previously described [4]. Dodecylsulfate -pol yacrylamide-gel electrophoresis was carried out in gel systems of the following composition: 0.1 M Tris-glycine buffer pH 8.6; 5 % acrylamide; 0.25 % N,N-methylene bisacrylamide; 0.05 % N,N,N',N'-tetramethylethylenediamine ; 0.08 % ammonium persulfate; 0.1 % sodium dodecylsulfate and 1.0 M urea.…”

Section: Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

“…It was possible to separate the two subunits by the action of denaturing agents [5], the denaturation being irreversible. A catalytically active subunit could be obtained only through degradation of the other subunit: isomaltase through mild alkaline treatment (which brought about an almost complete destruction of sucrase [4,6]) and sucrase through trypsin digestion (which destroyed almost completely isomaltase [7]). …”

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confidence: 99%

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Dissociation of Small‐Intestinal Sucrase. Isomaltase Complex into Enzymatically Active Subunits

Braun

Cogoli

Semenza

1975

European Journal of Biochemistry

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1 . The sucrase . isomaltase complex from rabbit small intestine dissociated into its subunits upon reaction with citraconic anhydride. They can recombine after deacylation under mild acidic conditions.2. When citraconylated, the subunits could be separated and isolated in a catalytically active form. 3. The previously reported procedure for separation of the subunits by alkaline treatment at pH 9.6 is apparently not due to contaminating degradative enzymes (possibly still present at undetectable levels in the isolated sucrase a isomaltase complex) but to the action of alkali.The sucrase . isomaltase complex is a membranebound protein which translocates some sugars across natural [I, 21 and artificial [3] membranes. It is a glycoprotein composed of two similar, but not identical subunits of molecular weight (M,) approximately 112000 [5], one splitting sucrose and the other isomaltose as well as a number of other a-glucosides. It was possible to separate the two subunits by the action of denaturing agents [5], the denaturation being irreversible. A catalytically active subunit could be obtained only through degradation of the other subunit: isomaltase through mild alkaline treatment (which brought about an almost complete destruction of sucrase [4,6]) and sucrase through trypsin digestion (which destroyed almost completely isomaltase [7]).The purpose of this paper is to present a procedure allowing the separation of both subunits of the complex and their isolation iil a catalytically active form. The procedure is based on reaction of the protein amino groups with citraconic anhydride. This is known to lead to dissociation of other oligomeric proteins [X-101 through a drastic change of the electrostatic interaction among subunits. Citraconylation of sucrase isomaltase complex followed by separation and deacylation under mild acidic conditions leads to catalytically active subunits. out before separation of the subunits reformation of the complex occurs.We also present additional evidence that in the mild alkaline treatment which leads to the preparation of isomaltase (as reported previously [4,6]) no action of contaminating proteases is involved. MATERIALS AND METHODSThe sucrase . isomaltase complex was prepared from rabbit small intestine as described [5,11] and sucrase and isomaltase activities were determined with the Tris -glucose-oxidase -peroxidase reagent [I 21 with sucrose and palatinose as respective substrates. The isomaltase activity will be expressed as palatinose units throughout. Protein was measured according to Lowry et al. [13], or from absorbance at 279 nm [11,14]. Free amino groups were determined with 2,4,6-trinitrobenzenesulfonic acid as described by Habeeb [I 51. In this determination samples of control enzyme subjected to the same conditions as the citraconylated enzyme were assayed at the same time. All results are expressed with respect to such controls.Neutral carbohydrates were released from the isomaltase subunit by treatment with 2 N H2S04 in evacuated tubes at 100 "C for 4 h. The ...

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Section: Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

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confidence: 99%

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Dissociation of Small‐Intestinal Sucrase. Isomaltase Complex into Enzymatically Active Subunits

Braun

Cogoli

Semenza

1975

European Journal of Biochemistry

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“…The rabbit small-intestinal sucrase-isomaltase complex has been isolated and characterized extensively in another laboratory [9][10][11][12]. The purification procedure is based primarily on the enzyme-substrate-like interaction with Sephadex, which results in an adsorption and retardation of the solubilized complex on Sephadex G-200 columns.…”

Section: Introductionmentioning

confidence: 99%

“…The same purification procedure has also been utilized for an immunochemical study of the human intestinal sucrase-isomaltase complex [ 13]. Recently the separation of free rabbit isomaltase after inactivation and separation of the sucrase moiety by incubation at pH 9.2 was reported [12,14].…”

Section: Introductionmentioning

confidence: 99%