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Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.

BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.

The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin. It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation. The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes. It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load. The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished. The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded. In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen. Extreme high loads are found to be able to divide single zones. As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients. For preparative gradient centrifugations the power of resolution will decrease with increasing sample load. A simple method to detect overloading in density gradient centrifugations is described.

The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.

Thermal fragmentation of the beta-galactosidase was studied in different buffer solutions and at different temperatures. Fragmentation of the subunits in small size polypeptides could be observed directly. The fragmentation proceeded in buffer solution, pH 7.0, at either 75 degrees C or 100 degrees C in the presence of sodium dodecyl sulfate. The elevated temperature appeared to accelerate this process. At 100 degrees C, pH 7.2, the fragmentation proceeded in the absence of sodium dodecyl sulfate, but in the presence of 8 M urea. Molecular weights, determined by sodium dodecyl sulfate disc gel electrophoresis were from 130,000 to about 20,000. Multiple bands were observed. After dissociation was complete at 37 degrees C, the fragments were purified by ion-exchange column chromatography. Of the five fragments thus obtained, four were homogeneous by disc-gel electrophoresis. Molecular weight of the homogeneous fragments were found to be near 25,000. The fifth comprised a mixture of four fragments having molecular weights from 29,000 to 72,000. Two of the fragments were active as the alpha-donor in in vitro complementation with mutant M15, which contains a deletion in the alpha-region of the z gene.

In nine patients with GM1 gangliosidosis, liver ganglioside GM1 beta-galactosidase (EC 3.2.1.23) activity ranged from less than 0.01% to 0.05% of normal. In a tenth patient's liver, much higher activity was found (0.5% of normal). In this patient the residual enzyme had the same molecular weight as beta-galactosidase A, the major form of beta-galactosidase of normal human liver. No activity was found that corresponded to beta-galactosidase B, the minor form of human liver beta-galactosidase. On starch gel electrophoresis, the patient's enzyme migrated less anodally than normal beta-galactosidase A, both before and after treatment with neuraminidase. Beta-Galactosidase from the patient had a Km that was higher then normal; 5-fold higher with ganglioside GM1 and 2-fold higher with 4-methylumbelliferyl beta-galactoside. The patient's enzyme crossreacted immunologically with normal beta-galactosidase A and had about 100-fold more antigenic activity per unit catalytic activity than the normal enzyme. The results indicate that in this patient a beta-galactosidase A protein with altered charge and altered catalytic properties was present in relatively normal amounts, the first electrophoretic variant reported for a patient with a lysosomal hydrolase deficiency.

Digestive enzymatic activities (disaccharidases, alkaline phosphatase, peptide hydrolases) have been determined in the mucosa of 14 patients with chronic pancreatitis. All had an abnormal secretin-pancreozymin test. Four patients had insulin-dependent diabetes mellitus, four a pathological glucose tolerance test. Nine patients had steatorrhoea. Maltase, sucrase, and alkaline phosphatase activity was significantly elevated in patients with exocrine pancreatic insufficiency, whereas those of lactase, trehalase, and peptide hydrolase were normal. Patients with steatorrhoea had higher maltase and sucrase activity than those without steatorrhoea, whereas decreased glucose tolerance had no effect on brush border enzymatic activity. It is suggested thatdecreased exocrine rather than decreased endocrine pancreatic function is responsible for the increase in intestinal disaccharidase and alkaline phosphatase activity, possible by the influence of pacreatic enzymes on the turnover of brush border enzymes from the luminal side of the mucosal membranes or by direct hormonal stimulation though cholecystokinin.

KB cells were synchronized by a double thymidine block procedure. An investigation was made of the activities of alpha-L-fucosidase (EC 3.2.1.51), alpha-D-galactosidase (EC 3.2.1.22), beta-D-galactosidase (ec 3.2.1.23), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), alpha-D-mannosidase (EC 3.2.1.24), beta-D-N-acetylgalactosaminidase (EC 3.2.1.53), and beta-D-N-acetylglucosaminidase (EC 3.2.1.52) from synchronized cultures, using appropriate artificial substrates. Ceramide glucosidase (EC 3.2.1.45) and ceramide trihexosidase levels (EC 3.2.1.47) were also investigated at various stages in the cell cycle, using appropriate glycosphingolipid substrates. Whereas each of these enzymes exhibited some activity throughout the cell cycle, peak activity (2- to 6-fold increase) occurred late in the S phase. Two molecular forms of ceramide glucosidase (optimal activity at pH 4.0 and pH 6.0) and two forms of ceramide trihexosidase (pH 4.0 and pH 7.5) were identified. Peak levels of the forms that preferred the relatively acid pH occurred earlier in the S phase of the cell cycle than those of the forms that were more active at the higher pH. The possibility that the forms with optimal activity at pH 4 are precursors of those with optimal activity at pH 6 to 7.5 is discussed. Precipitation of beta-galactosidase of synchronized KB cells with specific antibody revealed that changes in the activity of this enzyme during the cell cycle were the result of fluctuations in the amount of the enzyme.

Highly purfied preparations of the enzymes--yeast beta-fructofuranosidase, fungal beta-galactosidase and bacterial proteases have been isolated from crude preparations or culture liquids by adsorption on KMT microporous carboxyl cation exchanger. During desorption the enzyme activity has fully recovered and the specific activity increased 4.5-fold for beta-galactosidase and 54-fold for proteases.

Sphingomyelinase was obtained in excellent yield from liver and brain by homogenization with 0.05 M citrate-phosphate buffer, pH 4.5, containing 0.25% Triton-X-100 (v/v) followed by dialysis of the supernatant fluids against 1% glycine. Total recovery of enzyme was slightly less with tissue from Niemann-Pick disease compared with control tissue. Isoelectric focusing of liver and brain extracts was successfully used to resolve several species of sphingomyelinase. Three (I-III) of the five species were partially characterized. Enzyme I (pI 4.6) had a pH optimum of 4.8-5.0 in acetate buffer and a Km value of 0.026 mM. Both sphingomyelinases I and II were the major enzymes, whereas III, IV, and V were found at lower levels. Of the two major species in normal liver and brain (I and II), species I alone persisted in liver from the two cases of type C, while species III, IV, and V were present. In brain, only species II was decreased but the resolution of the brain enzymes was less satisfactory.

Adult rat brain beta-galactosidase was partially purified with the use of lactosylceramide, galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosyl-glucosylceramide, and 4-methylumbelliferyl theta-galactoside as substrates. Approximately 50-fold purification was achieved by solubilization, ammonium sulfate fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. Activities toward the above four substrates behaved essentially identically throughout the pruification procedure. Considerable differences were noted between the apparent properties determined with whole homogenate and those of the purer enzyme preparations. Based on these studies, assay procedures for the purified preparation for the three glycosphingolipid substrates were standardized. Inhibition studies with the use of varieties of simple sugars, oligosaccharide chains prepared from glycosphingolipids, and intact sphingolipids suggested that the enzyme which cleaves lactosylceramide may be different from the enzyme(s) which is active toward the other two glycosphingolipids. The oligosaccharide chains of the glycosphingolipids were much poorer inhibitors for the respective glycosphingolipid beta-galactosidases than the original intact glycosphingolipids or ceramide, and in some instances, even unrelated sphingolipids. These findings indicated the importance of the lipophilic groups and perhaps of the entire molecular configuration of glycosphingolipids in determining the specificity of these glycosphingolipid theta-galactosidases.

In previous studies, a cyanogen bromide peptide derived from amino-acid residues 3-92 of beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) was shown to have alpha-donor activity in intracistronic alpha-complementation. We have now isolated the defective beta-galactosidase alpha-acceptor protein from the deletion mutant strain M15 of Escherichia coli and find that it lacks residues 11-41 of betal-galactosidase. This is demonstrated by the isolation and sequence determination of a cyanogen bromide peptide from the M15 protein, which is identical to the corresponding peptide from beta-galactosidase except for the missing amino acids. We conclude that the alpha-donor peptide restores the region missing in the M15 protein.

Fungal beta-galactosidase was immobilized by covalent binding with KM-cellulose. The resultant preparation contained 3 mg protein per 1 g carrier; its specific activity was 65% of the initial one. As a result of immobilization pH optimum remained unchanged whereas the temperature optimum decreased from 65 degrees to 50 degrees. The seemings Km of the immobilized enzyme varied insignificantly as compared with Km of the soluble enzyme.

Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.

beta-d-phosphogalactoside galactohydrolase (beta-PGal) was isolated and purified from cell-free extracts of Streptococcus cremoris HP to apparent homogeneity to gel electrophoresis. Using the chromogenic o-nitrophenol-beta-d-galactopyranoside-6-phosphate as substrate, the purified enzyme exhibited a specific activity of 18.71 U/mg of protein and K(m) and V(max) values of 5.88 x 10(-4) M and 23.8 mumol of o-nitrophenol liberated per min per mg of protein, respectively. d-Galactose-6-phosphate was a weak competitive inhibitor of beta-PGal. Activity was relatively heat resistant and was maximal from pH 5.0 to 8.0 and over a temperature range of 45 to 52 C. Dithiothreitol, ethylenediaminetetraacetic acid, and citrate stimulated beta-PGal activity, whereas Mg(2+), Li(1+), and p-hydroxymercuribenzoate were inhibitory. Molecular weight of the enzyme was estimated at 6.76 x 10(4). Amino acid composition was similar to other beta-phosphogalactosidases previously investigated, with the exception that the S. cremoris enzyme contains a small amount of half cystine.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.

1. The p-nitrophenyl beta-D-galactosidase asctivity in rat liver homogenates of lysosome-rich fractions was shown to be markedly affected by the ionic composition of the medium. A stimulation of the reaction rate at pH 5 was produced by most of the salts tested, which contained anions such as acetate, SO4(2-) and Cl-, and cations such as Na+, K= and Mg2+. The most pronounced effect was observed with MgCl2. Only potassium glutamate was inhibitory. 2. Five peaks of beta-galactosidase activity obtained by DEAE-cellulose chromatography were equally sensitive to changes in the ionic composition of the medium. In the presence of added NaC1, the whole rate-pH curve was displaced towards higher pH values, the optimum being shifted from 2.0-2.5 to 3.5. The stimulation at pH 5.0 appeared to be mainly due to changes in Vmax., whereas the apparent Km was slightly modified. 3. Unlike the total, the free beta-galactosidase activity remained unchanged or even declined when KC1 was added to the reaction medium.

Pseudomonas sp. IFO-13309 and Actinobacillus sp. IFO-13310, bacteria which exhibit a symbiotic growth in a medium containing keratin sulfate as a sole carbon source, were isolated from soil. Extracts of these organisms were shown to contain an endoglycosidase, a sulfatase, and exo-beta-D-galactosidase, and an exo-beta-D-N-acetylglucosaminidase which, together, catalyze an extensive cleavage of corneal keratan sulfate. The Pseudomonas extract was particularly rich in the endoglycosidase activity and poor in the exoglycosidase activities. The Actinobacillus extract, in sharp contrast, contained principally the exoglycosidases. The sulfatase activity did not show this marked difference in distribution. A sulfatase was purified from the crude extract of Actinobacillus. The purified sulfatase reacted little or not at all with keratan sulfate, but acted on 2-acetamido-2-deoxy-6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose, and a tetrasaccharide trisulfate having 2-acetamido-2-deoxy-6-O-sulfo-D-glucose at the nonreducing end (prepared from keratan sulfate with an endogalactosidase). The enzyme removed one sulfate group from the tetrasaccharide trisulfate, producing an oligosaccharide which, unlike the parent oligosaccharide, was susceptible to hydrolysis with exo-beta-D-N-acetylglucosaminidase. The data suggest that the nonreducing end is the only site at wich enzymatic desulfation is carried out.

beta-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the beta-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg(-)) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg(-) and Deg(+) strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.

Lactase-phlorizin hydrolase (EC 3.2.1.-) has been purified from the monkey small intestine by gel filtration and ion-exchange chromatographic procedures and the properties of the purified enzyme complex have been studied. Lactose was the most active substrate. Cellobiose and other synthetic hetero-beta-glycosides were hydrolysed at a very much reduced rate. The rate of hydrolysis of phlorizin was about 2.5% that of lactose. Lactase and phlorizin hydrolase activities were indistinguishable by heat inactivation experiments. The purified enzyme complex also hydrolysed cerebrosides. Lactose hydrolysis was competitively inhibited by phlorizin as well as by the brain cerebroside. However, there was no mutual inhibition between phlorizin and the brain cerebroside. It is suggested that the native enzyme complex might have two catalytic sites, a phlorizin site and a cerebroside site but both hydrolysing lactose.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.

It has been previously reported that following neuraminidase treatment alpha-galactosidase A is converted into the B form, as revealed by electrophoresis. By a variety of techniques such as isoelectrofocusing, DEAE-chromatography and by enzyme kinetic parameters, no conversion of alpha-galactosidase A into B, or the reverse, could be detected after neuraminidase treatment. Only an apparent transformation of alpha-galactosidase A into B was revealed by Cellogel electrophoresis. In addition, a discrepancy was noticed between the pattern of electrophoretic migration on starch gel and Cellogel and the net electrical charges of the two alpha-galactosidases as deduced by isoelectrofocusing and DEAE-cellulose. Neuraminidase treatment did not affect the activity of alpha-galactosidase A towards the natural substrate, ceramidetrihexoside, but the activity of alpha-galactosidase B decreased by about 30% under the same conditions. The two forms of alpha-galactosidases A and B used in this study were extensively purified by classical procedures.

Sphingomyelinase was effectively extracted cultured fibroblasts homogenized in 1% glycine. The average specific activity was 24.6 nmol substrate hydrolyzed per hr per mg protein. Cultured cells from two cases of Niemann-Pick disease type A and one case of type B had markedly reduced enzyme activity, whereas, in type E cells, total activity was twice normal. Sphingomyelinase was resolved by isoelectric focusing into three peaks of activity (I-II), where I and II were the major forms. Species I had a pI of 4.6, II a pI of 4.8, and III a pI of 5.0. All three peaks, although markedly reduced, were present in cell extracts from both types A and B. All species of enzyme in B cells were found at higher levels than in A cells. Cultured cells from a case of type E contained sphingomyelinase I at a level several fold higher than normal, species II was absent, whereas III was found at near normal levels.