scholarly journals Rat small-intestinal β-galactosidases. Kinetic studies with three separated fractions

1968 ◽  
Vol 110 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Nils-Georg Asp ◽  
Arne Dahlqvist

1. Three fractions of β-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3–4, and the third one had a more neutral pH optimum at 5·7. 2. The two ‘acid’ β-galactosidase fractions had considerably lower Km values for hetero β-galactosides than for lactose. The Vmax. values were similar for all the substrates used (lactose, phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside). No difference could be detected between the two ‘acid’ fractions with respect to their enzymic properties (pH optimum, Km for the different substrates, Ki for lactose as an inhibitor of the hydrolysis of hetero β-galactosides, Ki for phenyl β-galactoside as an inhibitor of the hydrolysis of lactose, and relative Vmax. for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ‘neutral’ fraction had similar Km values for all the substrates hydrolysed, but with lactose as substrate the Vmax. was much higher than with the hetero β-galactosides. This fraction did not split phenyl β-galactoside or 6-bromo-2-naphthyl β-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero β-galactosidase activities of all the three fractions, and Ki for lactose as an inhibitor in each case was the same as Km for the lactase activity. Phenyl β-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero β-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.

1969 ◽  
Vol 114 (2) ◽  
pp. 351-359 ◽  
Author(s):  
Nils-Georg Asp ◽  
Arne Dahlqvist ◽  
Otakar Koldovský

1. Two β-galactosidases from human small-intestinal mucosa were separated by gel-filtration chromatography and the properties of the two enzymes were studied. Lactose and four hetero β-galactosides were used as substrates. 2. One of the enzymes was particle-bound and could be partially solubilized with papain. Of the substrates hydrolysed by this enzyme, lactose was hydrolysed most rapidly. This enzyme is thus essentially a disaccharidase and is named lactase. It is presumably identical with the ‘lactase 1’ described earlier. 3. The other enzyme was mainly soluble and hydrolysed all artificial substrates used, whereas no lactase activity could be detected. This enzyme has therefore been designated hetero β-galactosidase. 4. p-Chloromercuribenzoate (0·1mm) inhibited the hetero β-galactosidase completely but did not influence the activity of the lactase. Tris was a competitive inhibitor of both enzymes. 5. The residual lactase activity in the mucosa of lactose-intolerant patients may be exerted by a small amount of remaining lactase as such, or possibly by a third enzyme with a more acid pH optimum.


1971 ◽  
Vol 121 (2) ◽  
pp. 299-308 ◽  
Author(s):  
Nils-Georg Asp

1. An acid β-galactosidase, optimum pH4.0–4.5, in the human small-intestinal mucosa was separated and characterized. 2. Autolysis of mucosal homogenates at acid pH inactivated the lactase and hetero β-galactosidase; the total activity of the acid β-galactosidase was only slightly depleted, but a greater proportion of the enzyme was solubilized by this treatment. 3. Separation on a Sephadex G-200 column revealed that the acid β-galactosidase could occur in at least three different forms, probably representing monomer, dimer and octamer or polymer of the enzyme. 4. The properties of the different forms of the acid β-galactosidase were studied with regard to pH optimum, Km, rate of hydrolysis of different substrates, and sensitivity to p-chloromercuribenzoate and tris as inhibitors. All these properties were the same for the different forms of the enzyme. 5. The acid β-galactosidase hydrolyses lactose as well as hetero β-galactosides and contributes to the lactase activity of intestinal biopsies also when measured at pH 6. This enzyme may therefore be responsible for a considerable part of the residual lactase activity found in lactose-intolerant patients.


1989 ◽  
Vol 257 (4) ◽  
pp. G616-G623 ◽  
Author(s):  
H. A. Buller ◽  
A. G. Van Wassenaer ◽  
S. Raghavan ◽  
R. K. Montgomery ◽  
M. A. Sybicki ◽  
...  

Lactase-phlorizin hydrolase, a small intestinal disaccharidase, has been considered mainly an enzyme important only for the hydrolysis of lactose. After weaning in most mammals lactase-specific activity falls markedly, and, functionally, adult mammals are considered to be lactase deficient. However, the persistence of low levels of lactase activity in adulthood has never been explained. In addition, it has been suggested that lactase-phlorizin hydrolase is associated with glycosylceramidase activity when the enzyme is prepared by column chromatography, but it is unclear whether this represents copurified activities or two catalytic sites on one peptide. The developmental patterns of lactase-phlorizin hydrolase and other disaccharidases were investigated in homogenates of total rat small intestine; lactase and several glycosylceramidases were measured in immunoprecipitates from these homogenates using a monoclonal antibody. The developmental pattern of total lactase activity showed a steady 2.3-fold increase to adult levels (specific activity decreased eightfold), whereas total phlorizin-hydrolase activity increased 10.7-fold (specific activity decreased threefold). As expected, levels of both total and specific sucrase and maltase activities increased during development. In lactating rats total lactase activity showed a significant increase compared with adult males. The developmental pattern of the enzyme activities for the glycolipid substrates was similar to that found for lactase, and the immunoprecipitated enzyme showed a 40- to 55-fold higher affinity for the glycolipids than for lactose. Galactosyl- and lactosylceramide inhibited lactose hydrolysis by 38%, without a competitive pattern, suggesting two different active sites for lactose and glycolipid hydrolysis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


1963 ◽  
Vol 41 (1) ◽  
pp. 2107-2121 ◽  
Author(s):  
B. M. Laws ◽  
J. H. Moore

The digestive enzymes amylase and maltase were studied in acetone-dried powders or homogenates of the pancreatic and small intestinal tissues and small intestinal contents obtained from chicks of various ages. The stability of pancreatic amylase, which was relatively low in 0.15 M sodium chloride, was increased markedly by the presence of 0.02 M barbiturate buffer. The pH optimum of pancreatic amylase (chloride-activated) was 7.0 whereas that of intestinal maltase was 6.9. High levels of pancreatic amylase activity were found in the newly-hatched chick but these levels decreased during the following 20 days and then remained constant. The contrast between the high amylase and low maltase activities in the contents of the small intestine suggested that molecules of maltose, formed by the hydrolysis of starch, were absorbed as such by the mucosal cells. It appeared that maltose could be absorbed with equal facility from all sections of the small intestine of the 10-day-old chick but in the older birds maltose absorption seemed to occur more readily from the upper small intestine than from the duodenum and lower small intestine. A modified method for the determination of maltase activity is described.


1987 ◽  
Vol 65 (8) ◽  
pp. 717-725 ◽  
Author(s):  
John F. Sebastian ◽  
Richard S. Hinks ◽  
Ralf V. Reuland

A variety of modifiers of carboxypeptidase A (CPA) have been investigated in an effort to understand the structural requirements of inhibitors and activators of peptidase activity. It is proposed that an understanding of the mechanism of action of reversible activators of the enzyme may bear on the long standing question of whether the detailed mechanism of peptidase activity is different from that of esterase activity. An analog of the activator 2,2-dimethyl-2-silapentane-5-sulfonate, 5,5-dimethylhexanoate, was found to be a competitive inhibitor of the CPA-catalyzed hydrolysis of benzoylglycyl-L-phenyl-alanine. The modifier 4-phenyl-3-butenoate (styrylacetic acid) was determined to be an activator. The sulfonates benzene-sulfonate, p-toluenesulfonate, phenylmethanesulfonate, 2-phenylethanesulfonate, and 3-phenylpropanesulfonate were all found to be activators.


1971 ◽  
Vol 122 (1) ◽  
pp. 71-77 ◽  
Author(s):  
D. R. Trentham

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD+-bound enzyme are too large to measure but are nevertheless consistent with the low Km values of these substrates.


1969 ◽  
Vol 112 (1) ◽  
pp. 51-59 ◽  
Author(s):  
R. C. Siddons

1. Disaccharidase activities of the small and large intestines of the chick were studied. 2. Homogenates of the small intestine readily hydrolysed maltose, sucrose and palatinose (6-O-α-d-glucopyranosyl-d-fructose), hydrolysed lactose slowly and did not hydrolyse trehalose and cellobiose. 3. Within the small intestine the disaccharidases were located mainly in the intestinal wall; the activity in the contents accounted for less than 5% of the total activity. 4. The disaccharidases were non-uniformly distributed along the small intestine, the activities being greatest in the middle section. 5. The disaccharidase activities increased with age between 1 and 43 days. 6. Homogenates of the large intestine and contents readily hydrolysed maltose, sucrose, palatinose and lactose and hydrolysed cellobiose and trehalose slowly. 7. The large-intestinal disaccharidases were located mainly in the contents. 8. Similar Km and pH optimum values were found for the maltase, sucrase and palatinase activities of the large and small intestines. 9. The lactase activity of the large intestine was markedly affected by diet and had different Km and pH values from the small intestinal lactase. 10. Low activities of intestinal disaccharidase were found in 12-day-old embryos and marked increases in the intestinal disaccharidases of the developing embryo occurred 2–3 days before hatching.


1963 ◽  
Vol 41 (10) ◽  
pp. 2107-2121 ◽  
Author(s):  
B. M. Laws ◽  
J. H. Moore

The digestive enzymes amylase and maltase were studied in acetone-dried powders or homogenates of the pancreatic and small intestinal tissues and small intestinal contents obtained from chicks of various ages. The stability of pancreatic amylase, which was relatively low in 0.15 M sodium chloride, was increased markedly by the presence of 0.02 M barbiturate buffer. The pH optimum of pancreatic amylase (chloride-activated) was 7.0 whereas that of intestinal maltase was 6.9. High levels of pancreatic amylase activity were found in the newly-hatched chick but these levels decreased during the following 20 days and then remained constant. The contrast between the high amylase and low maltase activities in the contents of the small intestine suggested that molecules of maltose, formed by the hydrolysis of starch, were absorbed as such by the mucosal cells. It appeared that maltose could be absorbed with equal facility from all sections of the small intestine of the 10-day-old chick but in the older birds maltose absorption seemed to occur more readily from the upper small intestine than from the duodenum and lower small intestine. A modified method for the determination of maltase activity is described.


1976 ◽  
Vol 153 (2) ◽  
pp. 321-327 ◽  
Author(s):  
S Sivakami ◽  
A N Radhakrishnan

The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with α-1,4 and α-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.


1980 ◽  
Vol 189 (3) ◽  
pp. 447-453 ◽  
Author(s):  
C G Knight

1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4–6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3–6.0, with the mean value 7 × 10(-9) M.


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