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PubMed1992

Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.

1-Deoxynojirimycin↗
PubMed1990

Effect of the microbial lactase (EC 3.2.1.23) activity in yoghurt on the intestinal absorption of lactose: an in vivo study in lactase-deficient humans.

Breath hydrogen excretion was measured in eight lactase (EC 3.2.1.108)-deficient volunteers ingesting 18 g lactose in the form of milk, yoghurt and heated yoghurt. Total excess hydrogen excretion (area under curve) was significantly lower after yoghurt and heated yoghurt, than after milk: 103 (SE 29), 191 (SE 32), and 439 (SE 69) respectively (P less than 0.001). The oro-caecal transit time of fermentable components from yoghurt and heated yoghurt (mainly lactose) was longer than that from milk: 165 (SE 17), 206 (SE 19), v. 103 (SE 19) min (P less than 0.01). An intestinal perfusion technique was used in the same subjects after ingestion on two consecutive days of 18 g lactose in yoghurt and heated yoghurt. Significantly less lactose was recovered from the terminal ileum after yoghurt than after heated yoghurt meals: 1740 (SE 260) v. 2825 (SE 461) mg (P less than 0.05), and approximately one-fifth of the lactase activity contained in yoghurt reached the terminal ileum. These findings indicate that more than 90% of the lactose in yoghurt is digested in the small intestine of lactase-deficient subjects and suggest that both the lactase activity contained in the viable starter culture and a slow oro-caecal transit time are responsible for this excellent absorption.

Adult↗
PubMed1991

Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

Lactase-phlorizin hydrolase (LPH) splits lactose in the small intestine. LPH activity is high in the suckling; in many human populations the activity declines in adults, leading to adult-type hypolactasia, whereas in other populations the high LPH activity persists in adults. In the present work, we compared LPH sequences at the gene and cDNA level among adult subjects with high and low LPH activity. The complete intron-exon organization, including the sequences of all 17 exons and of the borders of all introns (as well as about 1,000 bp of 5' flanking region), was established for the cloned chromosomal LPH gene of a subject with persistence of lactase. Using PCR, we directly sequenced the exons of a hypolactasic subject. Except for silent mutations and the unknown linkage phase at two heterozygous positions, both coding sequences were identical. We further examined the LPH mRNA of a hypolactasic subject by S1 mapping and by sequencing a set of overlapping PCR products produced from cDNA templates. Except for allelic differences, the LPH sequence of the hypolactasic subject was identical to that of the LPH cDNAs of three subjects with persistence of lactase (one cDNA isolated previously by cloning and two characterized in the present work by PCR). No allele was peculiar to the hypolactasic subject. We conclude that humans with high or low levels of lactase can code for identical LPH enzymes.

Amino Acid Sequence↗
PubMed1988

Efficacy of addition of exogenous lactase to milk in adult lactase deficiency.

The efficacy of lactase by Kluyveromyces lactis in hydrolyzing milk lactose and reducing milk intolerance symptoms was tested in 52 proved lactose malabsorbers. The enzyme was added to milk administered to the patients, and H2 breath excretion (as an index of carbohydrate malabsorption), was determined by gas chromatograph technique, and milk intolerance symptoms were recorded. H2 mean excretion was 78.3 +/- 5.49 ppm after administration of intact whole milk 500 ml (test A), 43.5 +/- 4.99 ppm when lactase 2000 U was added to milk 500 ml immediately before administration (test B); 36.7 +/- 5.01 ppm when milk 500 ml was incubated for 12 h with lactase 1000 U (test C), and 29.7 +/- 4.35 ppm when the incubation was prolonged for 24 h (test D). Symptoms score was: test A = 5.85 +/- 0.56, test B = 3.71 +/- 0.45, test C = 2.77 +/- 0.63, test D = 1.7 +/- 0.68. A correlation index of r = 0.44 (p less than 0.01) was obtained between reduction in H2 mean excretion and reduction in symptoms score of a single individual. The addition of this lactase to milk seems to be effective in correcting lactose malabsorption, thus representing a convenient approach in milk intolerance.

Adult↗
PubMed1999

Species differences in the sites of cleavage of pro-lactase to lactase supports lack of selective pressure.

The pro-sequences in pro-lactase-phlorizin hydrolase (LPH) are needed for lactase to proceed past the ER, but are irrelevant as to the enzymatic activities. Hence, in all species removal of the pro- sequences (or most of them) must take place after the ER. Contrary to this, the details of the removal of these pro-sequences are to be expected to differ in the various species, since they are not subjected to selective pressure. Using site-directed mutagenesis we investigated processing in rabbit. The first cleavage occurs by furin (or furin-like PCs) and takes place at R-A-A-R(349) in the pro-sequence, generating the known 180 kDa intermediate. Replacing R(349) by Q results in a mutant which is not cleaved but nevertheless transported to the cell surface as demonstrated by immunofluorescence. Further processing of either the 180 kDa intermediate or the mutant is not directly mediated by furin-like PCs, but involves (also) other proteases. These results demonstrate that formation of the 180 kDa intermediate, consistently found only in rabbits, but not in man, is not essential for lactase transport: in all likelihood lack of selective pressure has led to species-specific processing of pro-LPH.

Animals↗
PubMed2005

Mutations in the translated region of the lactase gene (LCT) underlie congenital lactase deficiency.

Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. We initially assigned the CLD locus by linkage and linkage disequilibrium on 2q21 in 19 Finnish families. Here we report the molecular background of CLD via characterization of five distinct mutations in the coding region of the lactase (LCT) gene. Twenty-seven patients out of 32 (84%) were homozygous for a nonsense mutation, c.4170T-->A (Y1390X), designated "Fin(major)." Four rare mutations--two that result in a predicted frameshift and early truncation at S1666fsX1722 and S218fsX224 and two point mutations that result in substitutions Q268H and G1363S of the 1,927-aa polypeptide--confirmed the lactase mutations as causative for CLD. These findings facilitate genetic testing in clinical practice and enable genetic counseling for this severe disease. Further, our data demonstrate that, in contrast to common adult-type hypolactasia (lactose intolerance) caused by a variant of the regulatory element, the severe infancy form represents the outcome of mutations affecting the structure of the protein inactivating the enzyme.

DNA Mutational Analysis↗
PubMed1976

[Azoindoxyl methods for the histochemical investigation of hydrolases. I. Lactase (lactase-beta-glucosidase complex) (author's transl)].

An azoindozyl method for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) is described. The incubation medium consists of 5 mg 5-Br-4-Cl-3-indolyl-beta-D-fucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.02 ml hexazotized prosaniline in 10 ml 0.1 M citric acid phosphate buffer, pH 6-6.5. By means of this method lactase can be exactly localized in the brush border of the enterozytes in the jejunum of suckling rats. Compared to the corresponding indigogenic method the azoindoxyl reaction proceeds faster and the reaction product is often precipitated more precisely.

Animals↗
PubMed1987

Differences between lactase deficient and non-lactase deficient women with spinal osteoporosis.

Both osteoporosis and lactase deficiency are seen commonly in the United States. Since the latter may lead to avoidance of calcium sources and may exacerbate the bone disease in populations at risk, we studied lactose tolerance and histomorphometrically analyzed undecalcified transiliac bone biopsies in a consecutive group of postmenopausal women with the osteoporotic spinal compression fracture syndrome. Oral lactose tolerance tests prior to the biopsy clearly separated two groups. Sixty-five percent had abnormal test results. The bone biopsies in the lactase deficient group showed more osteoid volume and osteoid seam widths on examined trabecular bone. Analysis of tetracycline-labeled bone revealed significant increases in both single, double, combined single and double tetracycline labels, and the percent osteoid labeled with tetracycline. There was no difference in the calcification rates. These findings indicate different mineralization activity in lactase deficient patients, possibly reflecting their lower dietary calcium intake.

Biopsy↗
PubMed2000

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Animals↗
PubMed1975

Suitability of the azocoupling reaction with 1-naphthyl-beta-D-glucoside for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) in human enterobiopsies.

The suitability of the simultaneous azocoupling reaction with 1-naphthyl-beta-D-glucoside and hexazonium-p-rosanilin in the detection of the activity of lactase (or lactase-beta-glucosidase complex) in jejunal biopsies of patients with various forms of the malabsorption syndrome was tested. Results were compared with those obtained with the indigogenic method using 4-Cl-5-Br-3-indolyl-beta-D-fucoside which is the method of choice. Both methods gave identical results as far as the relative intensity of the brush border staining was concerned. The azocoupling method applied in unfixed cold microtome sections can be recommended for the routine diagnostics of the malabsorption syndrome when the indolyl substrate is not available.

Adult↗