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PubMed1983

Respiratory excretion of hydrogen and methane in Italian subjects after ingestion of lactose and milk.

Lactose malabsorption, by the breath hydrogen test, and lactose intolerance (presence of symptoms) were studied in twenty healthy Italian subjects after intake of 12.5, 25 and 50 g lactose, whole milk and low-lactose milk. A rise in respiratory concentration of hydrogen (greater than 20 ppm) (malabsorption) was found in fifteen subjects after 50 g lactose, in thirteen after 25 g and in seven after 12.5 g. Symptoms generally occurred in subjects presenting a rise in respiratory hydrogen excretion, but such a rise was often observed without symptoms. Thirteen subjects presented symptoms after 50 g lactose, but only three after 25 g and one after 12.5 g. Whole milk (500 ml) gave a lower incidence of lactose malabsorption than 25 g lactose (7/20 versus 13/20, P less than 0.05) and more subjects developed symptoms (7/20 versus 3/20, NS). Low-lactose milk produced no malabsorbers and one intolerant. Breath methane was detected constantly in seven subjects and in three on some of the days of observation. Respiratory methane excretion generally appeared to be unrelated to lactose ingestion.

Adult↗
PubMed2005

The molecular basis of lactose intolerance.

A staggering 4000 million people cannot digest lactose, the sugar in milk, properly. All mammals, apart from white Northern Europeans and few tribes in Africa and Asia, lose most of their lactase, the enzyme that cleaves lactose into galactose and glucose, after weaning. Lactose intolerance causes gut and a range of systemic symptoms, though the threshold to lactose varies considerably between ethnic groups and individuals within a group. The molecular basis of inherited hypolactasia has yet to be identified, though two polymorphisms in the introns of a helicase upstream from the lactase gene correlate closely with hypolactasia, and thus lactose intolerance. The symptoms of lactose intolerance are caused by gases and toxins produced by anaerobic bacteria in the large intestine. Bacterial toxins may play a key role in several other diseases, such as diabetes, rheumatoid arthritis, multiple sclerosis and some cancers. The problem of lactose intolerance has been exacerbated because of the addition of products containing lactose to various foods and drinks without being on the label. Lactose intolerance fits exactly the illness that Charles Darwin suffered from for over 40 years, and yet was never diagnosed. Darwin missed something else--the key to our own evolution--the Rubicon some 300 million years ago that produced lactose and lactase in sufficient amounts to be susceptible to natural selection.

Animals↗
PubMed1975

Intolerance of eight ounces of milk in healthy lactose-intolerant teen-agers.

The ability of lactose-intolerant individuals to tolerate 8 ounces of milk was determined in healthy teen-agers. Thirty-two blacks were studied with 50-gm lactose tolerance tests. Nineteen (59%) had a flat blood sugar curve and 13 (39%) also developed bloating, cramps, loose stools, or diarrhea with the test. These latter 13 were defined as lactose-intolerant. Seven of the 13 lactose-intolerant teen-agers (54%) developed abdominal bloating and/or cramps after drinking 8 ounces of milk (half-pint). None had diarrhea. Eight were symptomatic with the equivalent amount of lactose (12 gm) while only one had symptoms with the monosaccharide components of lactose, glucose and galactose. The symptoms with milk and 12 gm of lactose were less severe than with the 50-gm tolerance test. A history of a prior awareness of milk intolerance was obtained from 11 of the 13 lactose-intolerant subjects. At least one half of lactose intolerant teen-agers might be expected to be symptomatic after drinking 8 ounces of milk without other food. Milk intolerance should be considered in the nutritional planning for teen-agers with special attention to members of population groups with a high prevalence of lactose intolerance.

Adolescent↗
PubMed1993

Respiratory methane excretion in children with lactose intolerance.

To evaluate the relationship between colonic methane production and carbohydrate malabsorption, we measured end-expiratory methane levels in 70 normal and 40 lactose-intolerant children. Time-dependent excretion of hydrogen and methane was determined every 30 min for 120 min following a fasting oral lactose challenge (2 g/kg). Mean breath hydrogen levels in normals (lactose-tolerant) equaled 3.7 parts per million (ppm) throughout the study, but increased to > 10 ppm by 60 min and remained elevated in lactose-intolerant subjects. Breath methane in normal children averaged 1.6 ppm from 0 to 120 min. In contrast, CH4 excretion by lactose-intolerant children averaged 5.1 ppm at 90 min; and, by 120 min levels increased significantly compared with control. Breath methane levels in lactose-intolerant subjects following a lactose load continued to increase, however, despite the coingestion of exogenous lactase in amounts calculated to result in complete hydrolysis of the disaccharide. These data demonstrate that lactase-deficient children manifest significant increases in breath methane excretion following lactose ingestion and that enhanced methane production may be a consequence of several factors, including altered fecal pH and increased methanogenic substrates provided by colonic lactose fermentation. Further studies are required to determine the clinical significance of elevated methane production in lactose intolerance.

Breath Tests↗
PubMed2000

Improved lactose digestion and intolerance among African-American adolescent girls fed a dairy-rich diet.

OBJECTIVE: To determine whether African-American adolescent girls who were fed a dairy-rich diet for 21 days could adapt to lactose, experiencing an overall improvement in lactose tolerance as well as a decrease in hydrogen gas production. DESIGN: Twenty-one-day dietary intervention study. SUBJECTS/SETTING: Seventeen of 21 African-American girls (aged 11 to 15 years) enrolled in a calcium metabolism study chose to participate in the lactose tolerance study. Subjects were screened for any diseases, conditions, or medications that might alter calcium metabolism or colonic fermentation. Subjects were housed in a fraternity on the Purdue University, West Lafayette, Ind, campus, and were supervised 24 hours a day. INTERVENTION: Subjects consumed a dairy-based diet averaging 1,200 mg calcium and 33 g lactose per day for 21 days. Lactose digestion was assessed by an 8-hour breath hydrogen test on days 1 and 21, and symptoms of intolerance (abdominal pain, bloating, flatulence, and diarrhea) were evaluated hourly on a ranked scale during the breath hydrogen tests and once each evening during the 21-day feeding period. MAIN OUTCOME MEASURES: A comparison of breath hydrogen production and gastrointestinal symptoms at the beginning and end of the study. STATISTICAL ANALYSES PERFORMED: The Wilcoxon signed ranks test was used to compare the area under the curve for the 2 breath hydrogen tests. Spearman's p test for trend was used to determine whether there was a change in symptoms. All statistical analyses were 2-tailed and significance was set at P = .05. RESULTS: Fourteen of the 17 subjects had lactose maldigestion. Breath hydrogen excretion decreased significantly (P < .03) from the beginning (148.3 +/- 27.0 ppm x hours) to the end (100.7 +/- 19.3 ppm x hours) of the 21-day period. Gastrointestinal symptoms were negligible during both the breath hydrogen tests as were symptoms during the 21-day period. APPLICATIONS/CONCLUSIONS: The diet was well tolerated by the subjects. Furthermore, the decrease in breath hydrogen suggests colonic adaptation to the high-lactose diet. The results indicate that lactose maldigestion should not be a restricting factor in developing adequate calcium diets for this population. The existence of lactose maldigestion does not result in lactose intolerance in this population when it is fed a dairy-rich diet.

Adolescent↗
PubMed1975

Lactose and milk intolerance: clinical implications.

We studied 166 hospitalized male patients to determine the clinical importance of tolerance-test-determined "lactose intolerance," assumed to affect most of the world's adults. Abnormal lactose tolerance tests were found in 81% of 98 blacks, 12% of 59 whites of Scandinavian or Northwestern European extraction, and three of nine non-European whites. Seventy-two per cent of the "lactose-intolerant" subjects had previously realized that milk drinking could induce abdominal and bowel symptoms. Two hundred and forty milliliters of low-fat milk produced gaseousness or cramps in 59% of 44 "lactose-intolerant" men, and 68% were symptomatic with the equivalent amount of lactose. None of 18 "lactose-tolerant" men noted symptoms with milk or lactose. Refusal to drink 240 ml of low-fat milk served with meals correlated significantly with "lactose-intolerance": 31.4% versus 12.9% among "lactose-tolerant" patients. "Lactose intolerance" is common in adults and is a clinically relevant problem.

Adult↗
PubMed2003

Lactose digestion in pregnant African-Americans.

OBJECTIVE: This paper reports on the status of lactose digestion during early and late pregnancy and at 8 weeks postpartum in an African-American population. The hypothesis is that lactose digestion and milk tolerance do not change throughout pregnancy and do not differ from those of non-pregnant African-American women. DESIGN AND SUBJECTS: This longitudinal study determined lactose digestion after ingesting 240 ml of 1% fat milk containing 12 g of lactose at: (1) early pregnancy, prior to 16 weeks (n=148); (2) late pregnancy, 30-35 weeks (n=77); and (3) 8 weeks postpartum (n=93). One hundred and one comparably matched non-pregnant African-American women served as controls. RESULTS: Prevalence of lactose digestion, as measured by breath hydrogen, was 80.2% in the control women, 66.2% in early pregnancy, 68.8% in late pregnancy and 75.3% postpartum. The prevalence of women reporting symptoms was approximately 20% regardless of lactose absorption status. However, the control women reported significantly more symptoms than did the pregnant women. CONCLUSIONS: This study indicates that there is no significant change in lactose digestion during pregnancy. The prevalence of lactose intolerance for the pregnant African-American women studied is similar to that for non-pregnant African-American women and similar to previous prevalence reports in adult African-Americans. There was no change in the tolerance of lactose noted during pregnancy in these women. There were, however, fewer symptoms reported by the lactose-maldigesting pregnant women.

Academic Medical Centers↗
PubMed1975

Effect of dietary lactose on the absorption of protein, fat and calcium in the postweaning rat.

To assess the effect of dietary lactose on the intestinal absorption of protein, fat and calcium in lactose intolerance due to low levels of intestinal lactase, balance studies were carried out on postweaning rats (initial weight 164 g) for a period of 10 days. The experimental diets contained either 10% or 30% lactose, while in the control diets the lactose was replaced with equivalent amounts of sucrose. The excretions of fecal and urinary nitrogen, calcium and fat are expressed as precent of intake over the 10-day period. The fecal excretion of nitrogen and fat was significantly higher in the lactose groups (P less than 0.05). The fecal calcium excretion, however, was lower in the lactose groups, the difference being significantly only in the case of the 30% lactose diet (P less than 0.001). There was no difference in urinary nitrogen excretion between the lactose and sucrose groups, but urinary calcium excretion was significantly higher in the rats fed the 30% lactose diet (P less than 0.01). These findings suggest that dietary lactose may reduce the absorption of protein and fat, but not of calcium, in individuals with low levels of intestinal lactase activity.

Animal Nutritional Physiological Phenomena↗
PubMed1976

Lactose malabsorption in Mexican-American children.

Inability to absorb lactose due to low intestinal lactase is common in many population groups. This study is the first to compare lactose tolerance in 282 Mexican-American (MA) children and 51 Anglo-American (AA) children 2 to 14 years of age with the dietary intake of selected nutrients found in milk. A lactose tolerance test and a 24-hr dietary recall were obtained for each child. Gastrointestinal symptoms were carefully recorded for a 24-hr period following the lactose load. Overall prevalence of lactose malabsorption was 37% in MA children and 8% in AA children, and it increased with age. Number of symptoms occurring in lactose malabsorbers of both ethnic groups also increased with age. Mean protein intake exceeded Recommended Dietary Allowances at all ages for both ethnic groups. Mean consumption of vitamin A, calcium, and energy was below the Recommended Dietary Allowance for MA children. There were no differences in calories, nutrient, or milk intakes between lactose absorbers and malabsorbers, but AA children drank more milk than MA children. Fifteen percent of lactose-absorbing MA, 23% of malabsorbing MA, but no AA children reported having symptoms after drinking milk. There was a significantly greater incidence of lactose intolerance in MA as compared to AA children. This suggests that Mexican-Americans share in the high incidence of primary lactose intolerance characteristic of the majority of the orld's peoples.

Adolescent↗
PubMed1970

Incidence and clinical significance of lactose malabsorption in ulcerative colitis and Crohn's disease.

The incidence of lactose malabsorption was investigated in 85 patients with ulcerative colitis and 71 patients with Crohn's disease by means of lactose tolerance tests and disaccharidase determinations in small intestinal mucosa. Eight patients with ulcerative colitis (9%) and four with Crohn's disease (6%) had lactose malabsorption. A control group displayed a similar incidence. It is concluded that lactose malabsorption is not particularly common in ulcerative colitis and Crohn's disease. If it is present, its aetiology seems to be unrelated to the intestinal disease. Transitional lactose malabsorption was detected in two cases during a relapse of ulcerative colitis. Institution of a lactose-free (or lactose-poor) diet was an important supporting measure in seven patients who were unaware of their milk intolerance, in particular in two with ileostomy. Therefore, it is recommended that a lactose tolerance test should always be performed in patients with ulcerative colitis or Crohn's disease.Twenty-one patients with ulcerative colitis and nine with Crohn's disease, none of whom had lactose malabsorption, were placed on milk-free diets. A beneficial effect was noticed in five of the patients with ulcerative colitis, and in three of those with Crohn's disease. The mechanism is unknown. Evidence is presented that milk allergy is not responsible for the beneficial effect of a lactosefree diet in patients with associated lactose malabsorption.

Adolescent↗
PubMedDate not supplied

[Breath hydrogen test to evaluate lactose absorption and small bowel bacterial overgrowth in children].

The aim of this study was to determine the lactose absorption capacity and possible existence of bacterial overgrowth in the small bowel in asymptomatic school children of low social economic level in Marilia, a city located in the interior of São Paulo state. Eighty three children aging 7 to 15 years old without any gastrointestinal manifestations at least 30 days prior to the tests were studied. All the patients had fasted for at least 8 hours before the tests were performed. Lactose absorption was evaluated by breath hidrogen test after an overload of lactose 18 g in 10% aquous solution. Lactose intolerance was determined by the occurrence of clinical symptoms, such as diarrhea, abdominal pain, flatulence, etc in the following 24 hours after the test was performed. Bacterial overgrowth was evaluated by the breath hidrogen test after a 10 g lactulose load in aqueous solution. Lactose malabsorption was detected in 19 (22.9%) children and lactose intolerance was observed in 10 (12%) children. Lactose intolerance was more frequently observed in children who showed lactose malabsorption (6/19; 31.6%) than in those who presented a normal test (4/64; 6.3%) (P = 0.008). Bacterial overgrowth was detected in six (7.2%) children and showed no statistical relationship with lactose malabsorption. Ontogenetic lactose malabsorption verified in this group of school children is similar to the reported for Caucasian populations. Presence of bacterial overgrowth confirms the existence of asymptomatic environmental enteropathy in children of low social economic level.

Adolescent↗
PubMedDate not supplied

[Acute diarrhea: stool water loss in hospitalized infants and its correlation with etiologic agents and lactose content in the diet].

Forty weaned male infants were studied during their first year of life, all hospitalized with acute diarrhea in the Gastroenterology and Metabolism Unit of the Hospital "Umberto I", São Paulo, SP, Brazil. We evaluated and quantified water fecal losses, employing the metabolic bed technique, relating the feeding formula employed with the different causal enteropathogenic agents. 67.5% of the studied infants were under six months and 40% under three months of age. Two groups were randomly assembled to receive, lactose or lactose free feeding formulae. Twenty one patients received a lactose-containing formula (Ninho 10%) and the other 19 children were fed caseine (Portagen) formulae. According to coproculture results and identification of enteropathogenic agents, we divided the studied infants relating feeding formula with the presence or absence of the enteropathogenic Escherichia coli (EPEC): I-13 with positive coproculture for EPEC and diets which included lactose--(L/EPEC+); II--eight with negative coproculture for EPEC and diets which included lactose--(L/EPEC-); III--seven with positive coproculture for EPEC and lactose free diets--(G/EPEC+); IV--12 with negative coproculture for EPEC and lactose free diets. (G/EPEC-). The most frequently isolated agent at coproculture was EPEC, in 20 of the cases (50%), followed by Campylobacter (7.5%). It was also possible to observe that the frequencies of EIEC, Salmonella and Rotavirus were all equal (2.5%). Mixed infections occurred only between EPEC and EIEC, registering a frequency of 5%. The EIEC samples, associated to EPEC 0111 were serotyped as 0 28 ac: H- and 0 152:H-. The use of metabolic bed made the evaluation of fecal volumes possible by a simple and quick technique, thus allowing a closer clinical monitoring, as well as a more reliable evaluation of the patients hospitalized with acute diarrhea. Average acceptance volumes of the formulae--either with or without lactose--were always below the amount recommended by FAO/WHO (100 kcal/day) which shows the impact of acute diarrhea on the decrease of food intake. The average volumes of watery fecal losses found among any of the studied subgroups may be considered quite relevant when compared to standard values. Especially within the L/EPEC+ group fecal losses, both on the first day (83.56 ml/kg/day) and, mainly, on the second (119.44 ml/kg/day) reached exceedingly high levels indicating a disastrous association between the presence of EPEC in the small intestine and lactose offer in the diet. Thus, the results show that there exists a positive and significant association between poor lactose absorption and the presence of EPEC in the feces. WHO's recommendation proposing the use of diluted cow milk, in universal and indiscriminate administration, in the two first days of the disease, may represent a risk factor, not only for malnutrition, but also for the survival rates of children with severe diarrhea, especially those under six months of age and hospitalized with EPEC enteroinfection.

Acute Disease↗
PubMed1999

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose. I. The properties of two thermostable beta-glycosidases.

Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with SsbetaGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.

Chromogenic Compounds↗
PubMed2000

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases.

During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography. At 270 g/L initial lactose, they accumulate at a maximum concentration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of reaction and decreases directly proportional to increasing substrate conversion. Overall, CelB produces about 6% more hydrolysis byproducts than SsbetaGly. However, the product spectrum of SsbetaGly is richer in trisaccharides, and this agrees with results obtained from the steady-state kinetics analyses of galactosyl transfer catalyzed by SsbetaGly and CelB. The major transgalactosylation products of SsbetaGly and CelB have been identified. They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D-Galp-(1-->3)-lactose and beta-D-Galp-(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization.

Biotechnology↗
PubMed1984

A study of lactose absorption capacity in twins.

Lactose absorption capacity was determined by lactose tolerance tests with breath hydrogen determination in 102 healthy, adult, Hungarian pairs of twins in order to test monogenic Mendelian inheritance of the absorptive lactase phenotypes, lactose absorber and lactose malabsorber. Of the total, 52 pairs were monozygous (MZ) and 50 dizygous (DZ) twins of identical sex. All MZ twins were concordant with respect to lactase phenotype. Among DZ twins, the distribution of lactase phenotypes was in agreement with Hardy-Weinberg expectations derived from the frequencies of the hypolactasia gene in DZ and MZ twins, and in the general Budapest population. In the second part of the study, three commonly used methods of lactose tolerance testing, the blood glucose, the blood galactose, and the breath hydrogen tests, were compared in 49 pairs of twins concordant for lactase phenotype. Blood galactose concentration showed the greatest and only significant difference between the intrapair correlation coefficients of MZ and DZ, and no overlap between lactose absorbers and lactose malabsorbers. The intrapair correlation coefficients of peak breath hydrogen concentration in MZ and DZ twins did not significantly differ from zero, but the resolution of lactase phenotypes was satisfactory. Differences in glucose absorption and concentration in lactose absorbers and malobsorbers overlapped considerably, and among lactose absorbers correlation coefficients in DZ were higher than in MZ twins. In MZ and DZ twins, the difference in concordance and constancy of lactose intolerance symptoms was not significant.

Adolescent↗
PubMed1990

Modes of lactose uptake in the yeast species Kluyveromyces marxianus.

Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis. The strains fell into three groups. Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.

Biological Transport↗
PubMed1997

[Lactose intolerance and consumption of milk and milk products].

The disaccharide lactose is present as a natural component of foods only in milk and dairy products. In the gastrointestinal tract, lactose is hydrolysed by the enzyme beta-galactosidase (lactase) into glucose and galactose. These components are absorbed. With the exception of the caucasian race, the lactase activity decreases in most people at an age of 4 to 6 years. Lactose intake can cause symptoms of bloating, flatulence, abdominal pain, and diarrhea due to the lactose reaching the large intestine. This phenomenon is called lactose intolerance. It is generally recommended to those persons that they refrain from the consumption of milk and dairy products. However, most lactose intolerant people are able to digest small amounts of milk. They can also consume cheese that contains no (hard and semi-hard) or only small amounts of lactose (present in only 10% of soft cheeses). These products are very important sources of calcium. Compared to milk, the lactose content of yogurt is usually lower by about one third. Studies during the last 10 years have shown that in spite of its lactose content yogurt is very well tolerated by lactose intolerant persons. This advantage is ascribed to the presence of living lactic acid bacteria in fermented dairy products which survive passage through the stomach and also to the lactase present in these products.

Animals↗
PubMed2000

Mutants of the lactose carrier of Escherichia coli which show altered sugar recognition plus a severe defect in sugar accumulation.

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K(m) for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal. All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.

Biological Transport↗