The topography of lactose synthesis.

1. At short incubation times, and under suitable osmotic conditions, the lactose synthesized by Golgi-derived vesicles of rat mammary gland is 85-90% particulate. Evidence is presented for its occlusion within the lumen of the vesicles. 2. Ovalbumin is used as a bulky active-site inhibitor to show that the active site of lactose synthase lies on the inner face of the Golgi membrane. 3. Phlorrhizin and phloretin inhibit lactose synthesis by such vesicles, indicating the presence of a glucose-transport system. 4. The relationship of this topography to the synthesis of N-acetylneuraminyl-lactose and to the secretion of milk sugars is discussed.     

Pseudoreversion of lactose operator-constitutive mutants.

A set of pseudorevertants of lactose operator-constitutive (lacOc) mutant has been obtained. Analysis of a subset of these pseudorevertants indicates that, in some cases, the secondary mutation alters the lactose repressor (lacl gene product), whereas in others it seems to have occurred in the lactose operator (lacO) itself. Of the lacl gene mutations, the lacl8 mutation, already known to suppress all lacOc mutations nonspecifically, was recovered by a selection technique developed for this study. However, two additional lacl gene mutants were selected which appear to suppress lacOc sequences in a more-or-less specific fashion; repressor interaction with some operator sequences is facilitated, whereas the binding with lacO+ and others is attenuated concomitantly.     

Neurospora lactase. I. Properties of lactase preparations from a lactose utilizing and a lactose non-utilizing strain

Mutant strains of Neurospora which utilize lactose poorly as a carbon source are all found to form an enzyme with β-d-galactosidase activity. Purification and properties of β-d-galactosidase from a normal strain of Neurospora is described, and these properties are then compared with the properties of the β-d-galactosidase of a mutant strain which cannot utilize lactose as a carbon source. No differences between the two enzyme preparations were observed.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Cloning of chemically synthesized lactose operators.

Recombinant DNA molecules, constructed from the ColE1-Mk5 hybrid plasmid PMB9 and a chemically synthesized wild-type lactose operator segment, have been used to transform Escherichia coli. Up to 10% of the transformants (selected for the tetracycline-resistance property of PMB9) are partially constitutive for the lactose operon enzyme beta-galactosidase. In vitro studies demonstrate that these partially constitutive transformants contain plasmid DNA molecules which carry one or more lactose operators, and which will bind purified lactose repressor. Preliminary results with some modified operator sequences are also presented.     

Transport by the lactose permease of Escherichia coli as the basis of lactose killing.

Lactose killing is a peculiar phenomenon in which 80 to 98% of the Escherichia coli cells taken from a lactose-limited chemostat die when plated on standard lactose minimal media. This unique form of suicide is caused by the action of the lactose permease. Since uptake of either lactose or galactose by the lactose permease caused death, the action of rapid transport across the membrane must be the cause of the phenomenon. Alternative causes of lactose killing, such as accumulation of toxic metabolic intermediates or action of the beta-galactosidase, have been eliminated. It is proposed that rapid uptake of sugars by the lactose permease disrupts membrane function, perhaps causing collapse of the membrane potential.     

Lysis of Escherichia coli mutants by lactose.

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.     

The lactose synthetase particles of lactating bovine mammary gland. Preparation of particles with intact lactose synthetase

1. The particulate form of lactating bovine mammary lactose synthetase activity is shown to be more highly organized than previously reported. 2. A novel method of shattering frozen mammary tissue with effective cell disruption is described. 3. The apparent subcellular distribution of lactose synthetase was shown to reflect the method of homogenization. 4. After mild homogenization particles associated with a high content of intact lactose synthetase activity sedimented in the lysosome size range between 5×10⁴ and 3×10⁵g-min. 5. Lactose synthetase was dissociated and solubilized by VirTis homogenization and ultrasonic treatment. The activities and behaviour of UDP-galactose hydrolase, succinate dehydrogenase, β-glucuronidase and phosphodiesterase I were compared. 6. Inhibition of UDP-galactose hydrolase by UTP and α-lactalbumin was observed.     

Cloning of chemically synthesized lactose operators. II. EcoRI-linkered operators.

A 40 base, mainly duplex DNA segment, with the following sequence pAATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTT (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAAp (5') has been synthesized by combination of chemical and enzymatic methods. It consists of a wild-type lactose operator sequence (boxed) bracketed by "linker" sequences which permit excision of the segment from plasmid vehicles by the EcoRI restriction endonuclease. This segment has been ligated into the pMB9 plasmid and the resulting operator plasmids used to transform E. coli K-12. Among the transformant products were strains carrying plasmids with one, two, three, or four operator segments in tandem. Derepression of the lactose operon effected by these plasmids in vivo as well as the lifetimes of complexes formed between repressor and these plasmids in vitro increase with increasing numbers of operators per plasmid.     

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.     

Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient.

The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.