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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.

1. The influence of pH and the kind of buffer on the hydrolysis of lactose and four hetero-beta-galactosides (phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) by homogenates of rat small-intestinal mucosa has been studied. 2. There are at least two beta-galactosidases present in the homogenates, one with optimum pH3-4 and another with optimum pH5-6. 3. The enzyme with the lower pH optimum is mainly a heterogalactosidase. It hydrolyses lactose slowly. The other enzyme is mainly a disaccharidase, since it hydrolyses lactose much more rapidly than the heterogalactosides. 4. Under the conditions used, citrate had an inhibitory effect on the 6-bromo-2-naphthyl beta-galactosidase activity at pH3-4, but did not influence the 6-bromo-2-naphthyl beta-galactosidase activity at pH5-6 or the hydrolysis of the other substrates at any pH.

The activities of brush border lactase, lysosomal acid β-galactosidase, and cytoplasmic hetero-β-galactosidase have been measured in jejunal biopsies from 26 Zambian African subjects. All of them had adult hypolactasia. A low activity of brush border lactase was demonstrated, and that amounted to 5 to 90% (mean 62) of the total lactase activity at pH 6.0. Acid β-galactosidase and hetero-β-galactosidase activities were not decreased. Oral lactose tolerance tests were abnormal in 25 of the subjects, and 21 had gastrointestinal symptoms. There was a significant correlation between brush border lactase activity and the maximum blood glucose rise after oral lactose (P

An improved method for the assay of nmole quantities of phenol with a 4-aminoantipyrine reagent is reported and the use of the method for phenylglycosidase assay is shown. Tissue homogenate, serum, and different buffers did not interfere with the assay, thus making protein precipitation unnecessary.

A newly synthetized substrate, 2-naphthyl β-galactoside, was tested for the assay of human small-intestinal β-galactosidase activity. The substrate is hydrolyzed exclusively by the acid β-galactosidase and can, therefore, be used for the specific assay of that enzyme. A method for assay of enzyme activity is described. Liberated 2-naphthol is detected fluorometrically.

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.

1. Using a double-lumen tube perfusion system, solutions of lactose (50, 125 and 250 mmol/l) were introduced into the upper jejunum of six Zambian African subjects. By reference to a non-absorbable marker, polyethylene glycol, mol. wt 4000, the rates of absorption of lactose from each solution were calculated for a 300 mm jejunal segment.2. In three subjects total lactase activity of the jejunal mucosa and brush-border lactase and other disaccharidase activities were estimated. The jejunal total and brush-border lactase activities were low. Jejunal morphology was normal for African subjects.3. All subjects suffered abdominal colic and diarrhoea during and after the lactose perfusions. The kinetic curves for lactose were very shallow, and with all perfused solutions, there was a net movement of water into the jejunal lumen. The limited number of subjects, and the low and narrow range of enzyme activity, did not permit correlation between lactose absorption rate and lactase activity.4. In Zambian African subjects with adult hypolactasia, the jejunal mucosa absorbs a very small proportion of the perfused lactose.

1. An acid beta-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 beta-galactosidase; the total activity of the acid beta-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 beta-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 beta-galactosidase were studied with regard to pH optimum, K(m), 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 beta-galactosidase hydrolyses lactose as well as hetero beta-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.

1. Different forms of the rat small-intestinal acβ−galac→ssewereseparatedbyusingtheisoe≤ctric−focusingtechnique.Theisoe≤ctricp∮softhed⇔erentformswereatpH4.2,4.6,5.4,6.1and≈.8.2.Thetwoformsofacβ-galac→ssewereseparatedbyusingtheisoe≤ctric-focusingtechnique.Theisoe≤ctricp∮softhed⇔erentformswereatpH4.2,4.6,5.4,6.1and≈.8.2.Thetwoformsofacid' β-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had Kav. 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of `acid' β-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.

Specific methods are reported for the assay of three different β-galactosidases in small intestine mucosal biopsies. The specificity of the methods is evaluated by characterization of the enzymes in crude homogenates and by experiments in which the recovery of partially purified enzymes, added to mucosal homogenates from individuals with and without lactase deficiency, has been studied. Finally, the precision and the sensitivity of the methods have been evaluated from duplicate determinations in biopsy homogenates from subjects with and without lactase deficiency.

1. Three fractions of beta-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' beta-galactosidase fractions had considerably lower K(m) values for hetero beta-galactosides than for lactose. The V(max.) values were similar for all the substrates used (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside). No difference could be detected between the two ;acid' fractions with respect to their enzymic properties (pH optimum, K(m) for the different substrates, K(i) for lactose as an inhibitor of the hydrolysis of hetero beta-galactosides, K(i) for phenyl beta-galactoside as an inhibitor of the hydrolysis of lactose, and relative V(max.) for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ;neutral' fraction had similar K(m) values for all the substrates hydrolysed, but with lactose as substrate the V(max.) was much higher than with the hetero beta-galactosides. This fraction did not split phenyl beta-galactoside or 6-bromo-2-naphthyl beta-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero beta-galactosidase activities of all the three fractions, and K(i) for lactose as an inhibitor in each case was the same as K(m) for the lactase activity. Phenyl beta-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 beta-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.

A fluorometric method for the assay of intestinal lactase is described that is based on the assay of liberated galactose with galactose dehydrogenase. The method is more sensitive than the previously used glucose oxidase method. It is also free from the interference by high concentration of mucosal homogenate that can occur with the glucose oxidase method. The fluorometric procedure is therefore advantageous when extremely low levels of intestinal lactase activity are to be measured.

The “neutral” and “acid” β-galactosidases of rat small intestine can be measured separately when present in mixture. The “neutral” enzyme is measured in the presence of p -chloromercuribenzoic acid (CMB), which completely inhibits the “acid” enzyme. The “acid” enzyme can be measured at pH 3.5, at which pH the “neutral” enzyme has no activity. The accuracy of these methods has been tested by measurement of the activity of mixtures containing known amounts of both enzymes, and by measuring the K m for both enzymes in such mixtures, in crude homogenates containing both enzymes, and in isolated “neutral” and “acid” β-galactosidase preparations.