Improved purification of rat intestinal lactase

A rapid and improved method to obtain purified lactase from rat intestine is described. The purification procedure involved only two chromatographic steps. The degree of purification was far above (500 fold) the values reached with classical methods. Rabbit antisera raised to the purified lactase were characterized using conventional immunological techniques. The specificity of the lactase antibodies was confirmed by the lack of interference on maltase, aminopeptidase and alkaline phosphatase activities measured after papain extraction of the membrane proteins.     

Production of low-lactose milk by ectopic expression of intestinal lactase in the mouse mammary gland

We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creation of transgenic animals carrying a hybrid gene in which the intestinal lactase-phlorizin hydrolase cDNA was placed under the control of the mammary-specific alpha-lactalbumin promoter. Transgenic females expressed lactase protein and activity during lactation at the apical side of mammary alveolar cells. Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase synthesis in the mammary gland caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normally. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland to produce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestive enzyme into milk can selectively modify its composition.     

Further characterization of intestinal lactase/phlorizin hydrolase

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase.     

The nature of maturational decline of intestinal lactase activity

We have examined the nature of the decline of lactase (EC 3.2.1.23) activity in the maturing rat intestine. It was established in an initial study that the activity decline reflected a proportional reduction in the concentration of the enzyme protein. Accumulation patterns of label into lactase, total intestinal proteins and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) were compared, 4 h following administration of a tracer dose of [3H]leucine to weanling rats exhibiting a wide range of lactase decline. Accumulation of increasing amounts of label in total intestinal proteins and sucrase-isomaltase pools was found to accompany the lactase decline, in contrast to accumulation of a constant amount of label in the declining lactase pools. The pattern of increased label accumulation in total intestinal proteins was shown in a corollary study to reflect a corresponding acceleration of total protein synthesis. On this basis, the finding of a constant amount of label in the declining lactase pools suggested a constant synthesis of lactase. We proposed earlier that associated reductions in enterocyte life-span (leading to correspondingly less lactase accumulation) rather than suppressed synthesis may provide the primary causal basis of lactase decline in the postweaned mammal.     

Gene-dosage effect on intestinal lactase activity demonstrated in vivo.

The activities of the disaccharidases lactase, maltase, and sucrase were determined in upper jejunal biopsies of 65 healthy adult German males. The study was an attempt to demonstrate the gene-dosage effect on lactase activity expected from the presence of a "hypolactasia" (l) and a "lactase-persistence" (L) allele in the German population. In contrast to lactase/sucrase ratios, lactose/maltose ratios showed a trimodal distribution in proportions of presumed genotypes LL, Ll, and ll compatible with Hardy-Weinberg equilibrium. The frequency of homozygotes ll (13.8%) was similar to the average frequency of lactose malabsorbers in Germany reported in the literature. The importance of considering the lactase gene-dosage effect in population studies of lactase activity is discussed.     

Recessive inheritance of the adult type of intestinal lactase deficiency.

In order to investigate the genetic control of the adult type of intestinal lactase deficiency, 61 families with 177 children over 6 years of age were investigated. The results strongly suggest that this deficiency is inherited as a simple Mendelian recessive trait.     

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.     

High intestinal lactase concentrations in adult Arbs in Saudi Arabia.

The maximum rise in blood glucose after 50 g lactose by mouth was determined in 40 adult Arabs. Out of 30 Bedouin, urban Saudi, and Yemeni and 9 of mixed ancestry (usually partly African), 25 (83%) and 2 (22%) respectively showed an increase of over 1-1 mmol/1 (20 mg/100 ml). In common with most northern Europeans and Hamitic people of northern Africa, Arabs in Saudi Arabia usually have high intestinal lactase concentrations in adult life. This persistence of high levels probably originated in the Arabian peninsula. Its selective advantage may have been associated with the fluid and calorie content of camels'' milk, which is important for survival in desert nomads.