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PubMed2001

Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I.

The lactose synthase (LS) enzyme is a 1:1 complex of a catalytic component, beta1,4-galactosyltransferse (beta4Gal-T1) and a regulatory component, alpha-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to beta4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to beta4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on beta4Gal-T1, while the N-acetyl group-binding pocket in beta4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for lactose synthase.

Acetylglucosamine↗

PubMed1992

Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer.

The use of isopropyl-beta-D-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. The resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein.

Aphthovirus↗

PubMed1979

The biochemical and histochemical demonstration of lactase induction in fetal rat intestine by intra-amniotic injection of lactose.

A single dose of 20 mg beta-D-lactose injected into the amniotic sac of rats on day 17 of pregnancy induced an increase in lactase activity in fetal jejunum. This effect was first noted two days after injection and lasted for at least two additional days. Histoenzymatic investigation indicated that this enzyme was located on the surface of the absorptive cells lining the villi and thus corresponds to the "dietary" form of beta-galactosidase. A much smaller increase, based presumably on progressive increase in fetal size (age) was found in control fetuses which had received glucose or no injections. Peak lactase values in fetuses receiving lactose were substantially higher than peak values in control fetuses. In both lactose-injected and non-injected rats which were allowed to deliver, there was a sharp drop in lactase values coincident with birth.

Amnion↗

PubMed1978

The geographic hypothesis and lactose malabsorption. A weighing of the evidence.

Reviewed in this article is evidence bearing on the geographic hypothesis advanced eight years ago to explain the striking ethnic or racial differences in prevalence of primary adult lactose malabsorption that are found around the world. Most evidence is found to support the hypothesis and the likelihood that some human groups came to have low prevalences of such lactose malabsorption because of selective pressures over a long historical period that favored the adult lactose absorber under particular ecological conditions.

Adult↗

PubMed1984

Transport by reconstituted lactose carrier from parental and mutant strains of Escherichia coli.

The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lower Km but similar Vmax compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.

Bacterial Proteins↗

PubMed1989

The influence of ascorbic acid and lactose on the interaction of iron with each of cobalt and zinc during intestinal absorption.

The effect of ascorbic acid and lactose on the interaction between iron and each of zinc or copper during intestinal absorption was studied in normal and iron-deficient rats. It was found that addition of cobalt to the iron dose inhibited absorption of iron to 42% in normal rats and to 46.7% in iron deficient ones. The presence of zinc with the iron dose also inhibited absorption of iron to 34.6% in normal rats and to 32.2% in case of the iron deficient ones. The addition of ascorbic acid to the combined dose of Fe + Co enhanced absorption of iron by five times in normal and in iron deficient rats. In case of the combined dose of Fe + Zn the enhancement was four times in normal rats and six times in iron deficient ones. The addition of lactose to the combined dose of either Fe + Co or Fe + Zn corrected the inhibiting action of either cobalt or zinc on iron absorption. Based on these findings, it is recommended that ascorbic acid and lactose be added to therapeutic multimineral preparations.

Animals↗

PubMed1982

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice.

Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture.

Animals↗

PubMed2006

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture.

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l(-1)) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h(-1). Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h(-1). The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms.

Acetate Kinase↗

PubMed1996

The effect of cations on the hydrolysis of lactose and the transferase reactions catalysed by beta-galactosidase from six strains of lactic acid bacteria.

beta-Galactosidases from Lactobacillus delbruekii subsp. bulgaricus 20056, Lb. casei 20094, Lactococcus lactis subsp. lactis 7962, Streptococcus thermophilus TS2, Pediococcus pentosaceus PE39 and Bifidobacterium bifidum 1901 were partially purified. The rate of hydrolysis of lactose given by the predominant beta-galactosidase activity from each of the bacteria studied was in all cases enhanced by Mg2+, while the effect of K+ and Na+ differed from strain to strain. The beta-galactosidases from all strains also catalysed trans-galactosylation reactions. The types of oligosaccharides produced appeared to be very similar in each case, but the rates of their production differed. All the beta-galactosidases were also capable of hydrolysing galactosyl-lactose although, unlike the other bacteria studied, Lb. delbruekii subsp. bulgaricus 20056 and Lc. lactis subsp. lactis 7962 were unable to utilise galactosyl-lactose as a carbon source for growth.

Cations↗

PubMed2005

Identification of a lactose-responsive element upstream of the promoter of Bacillus megaterium beta-galactosidase-encoding gene mbgA.

The Bacillus megaterium mbgA gene encodes a lactose-hydrolyzing beta-galactosidase. An AraC/XylS-type activator BgaR can activate mbgA transcription in response to lactose. In this report, we show by various deletion analyses and point mutagenesis analyses that an inverted repeat centered at position -60.5 relative to the mbgA transcriptional initiation site is the cis-acting element responsible for lactose induction of mbgA expression.

Bacillus megaterium↗

PubMed2003

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier.

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7. The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4-7% of the original dry cell weight. The purified product, at a concentration of 12 g l(-1), formed emulsions that were stable for 3 months when subjected to a range of pH (3-11) and NaCl concentrations (2-50 g l(-1)). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4-6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.

Carbohydrate Metabolism↗

PubMed2006

Lactose-induced production of human soluble B lymphocyte stimulator (hsBLyS) in E. coli with different culture strategies.

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l(-1) and a productivity of 0.11 g l(-1) h(-1).

B-Cell Activating Factor↗

PubMed2006

Transient lactose malabsorption in patients affected by symptomatic uncomplicated diverticular disease of the colon.

Lactose malabsorption (LM) may be secondary to several small bowel diseases, and small intestinal overgrowth (SIBO) may be one of them. We looked for a correlation between symptomatic diverticular disease of the colon and LM and assessed whether this correlation may be related to SIBO. Ninety consecutive patients (pts; 39 males, 51 females; mean age, 67.2 years; range, 32-91 years) affected by symptomatic uncomplicated diverticular disease of the colon were evaluated to assess orocecal transit time (OCTT), SIBO, and LM by lactulose and lactose H2 breath test (H2-BT) at entry and after 8 weeks of treatment. OCTT was delayed in 67 of 90 pts (74.44%). Fifty-three of 90 pts (58.88%) showed SIBO, and OCTT was normal in 23 of 90 pts (25.56%). LM was diagnosed in 59 of 90 pts (65.55%): 49 of 59 (71.74%) were simultaneously affected by SIBO and delayed OCTT (and thus 49 of 53 pts [92.45%] with delayed OCTT and SIBO were affected by LM); 3 of 59 pts (5.09%) showed only delayed OCTT; 7 of 59 pts (11.86%) did not show either SIBO or delayed OCTT. The association of LM and SIBO was statistically significant (P < 0.001). Seventy-nine of 86 pts (91.86%) showed normal OCTT, while OCTT remained prolonged but shorter in the remaining 7 pts (8.14%). SIBO was eradicated in all pts completing the study, while a new lactulose H2-BT showed persistence of SIBO in one pt with recurrence of symptomatic diverticular disease. Forty-seven of 59 pts (79.66%) had a normal lactose H2-BT (P < 0.002), while 12 of 59 pts (20.34%) showed persistence of LM. LM disappeared in 46 of 49 pts (93.88%) concurrently with normalization of OCTT and eradication of SIBO (P < 0.002); it also disappeared in 1 of 3 pts (33.33%) previously affected by delayed OCTT (without SIBO) and LM concurrently with normalization of OCTT. On the contrary, it persisted in all pts with normal OCTT and absence of SIBO. Moreover, it persisted also in the pt with recurrence of symptomatic diverticular disease and persistence of SIBO. In conclusion, most pts affected by symptomatic uncomplicated diverticular disease of the colon showed LM, and in more than 70% of cases it disappeared after successful treatment of the colonic disease.

Adult↗

PubMed1989

Microanalysis of the metabolic intermediates of lactose synthesis in human milk and plasma using bioluminescent methods.

Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small sample volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters sample. For the assays of the metabolites, recoveries ranged from 91 to 107%. For sample sizes of 2 to 5 microliters of protein free sample, the detection limits for milk were G1P, 0.09 microM; G6P, 0.05 microM; UDPhexose, 0.07 microM; UDP-Glc, 0.03 microM; glucose, 9 microM; and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.

Animals↗

PubMed1982

Specific reactivity of lipid vesicles conjugated with oriented anti-lactose antibody fragments.

The method previously described (Sinha, D. and Karush, F. (1979) Biochem. Biophys. Res. Commun. 90, 554--560) for the oriented attachment of immunoglobulins to lipid vesicles has been used to confer specific reactivity on liposomes by their conjugation with anti-lactose Fab' fragments derived from rabbit IgG antibody. It is estimated that one-third of the Fab' fragments was irreversibly attached to liposomal membrane, resulting in a membrane concentration of 2 mmol of Fab' per mol of total lipid. The specific reactivity of the modified liposomes was demonstrated by agglutination with a multivalent, lactose-containing diheteroglycan. The availability of virtually all of the binding sites of the attached antibody for reaction with ligand was established by a fluorescence quenching titration with N-(N epsilon-Dnp-L-lysyl)-p-aminophenyl-beta-lactoside. An intrinsic association constant of 8.9 x 10(6) M-1 was found for the attached Fab' compared to a value of 2.8 x 10(6) M-1 for free anti-lactose Fab'. In addition the maximum values for the quenching by bound ligand of the fluorescence of free and attached antibody were the same. It can be concluded that the chemical procedures used to effect attachment of the antibody to the lipid vesicles allow retention of the original structure of the antibody site and its accessibility to external components.

Agglutination↗

PubMed1988

The lactose carrier of Klebsiella pneumoniae M5a1; the physiology of transport and the nucleotide sequence of the lacY gene.

A comparison has been made between the physiology and amino acid sequence of the lactose carriers of Klebsiella pneumoniae M5a1 and Escherichia coli K-12. The membrane transport of lactose was much weaker in Klebsiella than in E. coli. On the other hand o-nitrophenylgalactoside uptake by Klebsiella was distinctly greater than with E. coli. In spite of the differences in sugar transport between the two organisms, the amino acid sequences of the respective lactose carriers were remarkably similar (60% of the amino acids are identical).

Amino Acid Sequence↗

PubMed1996

Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli.

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.

Base Sequence↗

PubMed1984

Myristoyl esters of lactose.

Whereas lactose did not undergo a base-catalyzed transesterification with methyl esters of fatty acids, methyl beta-lactoside reacted under identical conditions to give mono- and di-myristates. This difference in behavior is explained in terms of the formation of an unreactive, internally chelated potassium-lactose complex. Supporting evidence for this hypothesis is the observed change in the anomeric equilibrium of lactose in the presence of potassium carbonate. The monomyristates of methyl beta-lactoside were assigned the structures of 3' and 6' derivatives, and it is concluded that the diesters are the 3',6', and 6,6' derivatives.

Chemical Phenomena↗