The Transmembrane Helices of Beef Heart Cytochrome Oxidase

The locations of the transmembrane helices in the 12 subunits of beef heart cytochrome oxidase were predicted with a modified form of the von Heijne-Blomberg hydrophobicity scale. Based on ~20 residues per transmembrane helix, about 480 of the estimated 660 helical residues (36.8% of 1,793 total residues) are expected to be in transmembrane helices that have their axes tilted by a small angle α from the normal to the plane of the membrane. This angle is calculated to be ~30°, based on the observed overall tilt angle θ of 39° obtained from circular dichroism (CD) measurements on multilamellar films, or about 25°, based on the observed tilt angle θ of 36° obtained from the infrared linear dichroism of films. For 21 residues per transmembrane helix, the calculated values of α become 32° and 28°, respectively, depending upon the value of θ used. Thus, a transmembrane helical tilt angle of ~30° accounts for the predicted transmembrane stretches in cytochrome oxidase if 20-21 residues are sufficient to span the membrane. Additional helical residues in the lipid head region may deviate by a larger angle from the normal to the plane of the membrane in cytochrome oxidase.     

Cold-Adapted β-Galactosidase from the Antarctic Psychrophile Pseudoalteromonas haloplanktis

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH₂-terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M_r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth

Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wall β-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since the pH optimum for the enzyme is about 5.0. It has been suggested that enhancement of β-galactosidase and/or other glycosidase activity mediates growth promotion by auxin or low pH. This hypothesis was tested by examining the effect of inhibitors of β-galactosidase and β-glucosidase. Severe inhibition of measureable β-galactosidase or β-glucosidase activity was found to have no effect on auxin- or acid-promoted growth. It is concluded that neither β-galactosidase nor β-glucosidase plays an important role in short term growth promotion by auxin or acid. The data do not rule out the possibility that some other cell wall glycosidase is involved in auxin or acid action.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

High Production of Thermostable β-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis

By cloning the β-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. β-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70°C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, β-galactosidase is suitable for application in industrial processes.     

Respiratory Chain of Colorless Algae II. Cyanophyta

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad α-bands near 560 mμ. At −190° these bands split to give 4 peaks in the α-region for b and c-type cytochromes, but no α-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mμ. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mμ. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.     

Inhibition of glycosidases by aldonolactones of corresponding configuration: The C-4- and C-6-specificity of β-glucosidase and β-galactosidase

1. In barley, β-glucosidase and β-galactosidase are separate enzymes. The former also displays β-d-fucosidase activity. 2. In the limpet, Patella vulgata, β-glucosidase activity is associated with the β-d-fucosidase, previously shown to be a separate entity from the β-galactosidase also present. 3. Almond emulsin presents all three activities as a single enzyme. Each is equally inhibited by glucono-, galactono- and d-fucono-lactone. 4. In rat epididymis, there is no significant β-glucosidase activity, nor is there appreciable inhibition of the β-galactosidase and β-d-fucosidase activities of the preparation by gluconolactone.     

beta-Galactosidases in Ripening Tomatoes

Tomatoes (Lycopersicon esculentum L.) contained a high level of β-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of β-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, β-galactosidases I and III levels decreased but the β-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K_m value with p-nitrophenyl-β-galactoside, and stability with respect to pH and temperature. β-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of β-1, 4-linked galactose. The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Host-Pathogen Interactions: I. A Correlation Between alpha-Galactosidase Production and Virulence

Resistance or susceptibility of Red Kidney, Pinto and Small White beans (Phaseolus vulgaris) to the alpha, beta, and gamma strains of Colletotrichum lindemuthianum was either confirmed or established. These fungal strains secrete α-galactosidase, β-galactosidase and β-xylosidase when grown on cell walls isolated from the hypocotyls of any of the above bean varieties. These enzymes effectively degrade cell walls isolated from susceptible 5-day old hypocotyls but degrade only slightly the walls isolated from resistant 18-day old hypocotyls. The amounts of the β-galactosidase and β-xylosidase secreted by the 3 fungal strains are relatively low and are approximately equivalent. The secretion of these 2 enzymes is not dependent upon the bean variety from which the hypocotyl cell walls used as a carbon source were isolated. However, the fungal strains secrete greater amounts of α-galactosidase when grown on hypocotyl cell walls isolated from susceptible plants than when grown on walls from resistant plants. Virulent isolates of the fungus, when grown on hypocotyl cell walls isolated from a susceptible plant, secrete more α-galactosidase than do attenuated (avirulent) isolates of the same fungal strain grown under the same conditions. The α-galactosidase secreted by each of the fungal strains is capable of removing galactose from the hypocotyl cell walls of each bean variety tested. Galactose is removed from the cell walls of each variety at the same rate regardless of whether the cell walls were isolated from a susceptible or resistant plant.     

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Inducible and Constitutive β-Galactosidase Formation in Cells Recovering from Protein Synthesis Inhibition

Inducible and constitutive β-galactosidase formation and radioactive amino acid incorporation were measured in cells recovering from various treatments which inhibit protein synthesis in the cell. Undelayed β-galactosidase formation was found in stringent auxotrophs recovering from amino acid starvation, in cells recovering from glycerol or potassium starvation, and in bacteria recovering from puromycin treatment. Delayed β-galactosidase formation was found in relaxed auxotrophs recovering from amino acid starvation and in prototrophs recovering from chloramphenicol or from tetracycline treatment. The length of this delay was directly proportional to the duration of the treatment. All cells recovering from the various treatments exhibited a slightly decreased rate of β-galactosidase formation and an increase in radioactive amino acid incorporation.     

The Role of beta-Galactosidases in the Modification of Cell Wall Components during Muskmelon Fruit Ripening

The changes in activities of soluble β-galactosidase and two forms of wall-bound β-galactosidases extracted with NaCl and EDTA were investigated throughout the development of muskmelon (Cucumis melo L. cv Prince) fruits. DEAE-cellulose ion-exchange chromatography of soluble β-galactosidase revealed the presence of two isoforms. Soluble isoform I was detected in all stages throughout the fruit development, whereas soluble isoform II appeared around 34 d after anthesis when fruit ripening initiated. Both NaCl- and EDTA-released β-galactosidase activities also increased as ripening proceeded. The soluble and wall-bound forms behaved differently upon ion-exchange chromatography. Enzymological properties such as optimum pH, optimum temperature, K_m values for p-nitrophenyl β-d-galactopyranoside, and inhibition by metal ions were nearly similar in all forms. Molecular sizes of pectic polymers and hemicelluloses extracted from fruit mesocarp cell walls were shifted from larger to smaller polymers during ripening, as determined by gel filtration profiles. NaCl-released β-galactosidase from cell walls of ripe fruits had the ability to degrade in vitro the pectin extracted from preripe fruit cell walls to smaller sizes of pectin similar to those that were observed in ripe cell walls in situ. Both soluble isoform I and II were able to degrade in vitro the 5% KOH-extractable hemicellulose from preripe fruit cell walls to sizes of molecules similar to those that were observed in ripe cell walls in situ. Soluble isoform I and the NaCl-released form from ripe fruits were able to modify in vitro 24% KOH-extractable hemicellulose from preripe cell walls to sizes of molecules similar to those that were observed in ripe fruits in situ.     

ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER : I. Biochemical Methods

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b₅ and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-β-glucosaminidase, β-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.     

Relation of glycosidases to bean hypocotyl growth

The enzymes β-glucosidase, α-glucosidase, β-galactosidase, α-galactosidase, and β-xylosidase were detected in Phaseolus vulgaris L. var. Red Kidney bean hypocotyl tissue throughout the first 13 days of development with p-nitrophenyl glycosides as substrates. Activities of all enzymes except β-glucosidase declined as a function of increasing tissue age. In contrast, β-glucosidase activity increased rapidly 3 days after imbibition to a maximal activity at 5 days and then subsided to one-third the maximum by day 7. This activity peak immediately preceded the logarithmic phase of hypocotyl growth. This enzyme is strongly associated with cell walls during extraction, suggesting that it is wall-bound in situ. Various polysaccharide substrates were used to evaluate the specificity of this enzyme.     

Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson''s disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson''s disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson''s disease and other neurodegenerative disorders.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

LAC4 Is the Structural Gene for β-Galactosidase in KLUYVEROMYCES LACTIS

Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac^- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac⁺ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the K_m for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac⁺ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.     

Inhibition of glycosidases by aldonolactones of corresponding configuration. The specificity of α-l-arabinosidase

1. The previous study (Conchie, Gelman & Levvy, 1967b) of the specificity of β-glucosidase, β-galactosidase and β-d-fucosidase in barley, limpet, almond emulsin and rat epididymis was extended to α-l-arabinosidase. 2. The inhibitory action of l-arabinono-(1→5)-lactone was tested against all four types of enzyme, and α-l-arabinosidase was examined for inhibition by glucono-, galactono- and d-fucono-lactone. 3. In emulsin, the enzyme that hydrolyses β-glucosides, β-galactosides and β-d-fucosides also hydrolyses α-l-arabinosides. Rat epididymis resembles emulsin except that, as already noted, it lacks β-glucosidase activity. 4. In the limpet, α-l-arabinosidase activity is associated with the enzyme that hydrolyses β-glucosides and β-d-fucosides, and not with the separate β-galactosidase. 5. The effects of the different lactones on the barley preparation suggest that α-l-arabinosidase activity is associated with the β-galactosidase rather than with the enzyme that hydrolyses β-glucosides and β-d-fucosides. Fractionation and heat-inactivation experiments indicate that there is also a separate α-l-arabinosidase in the preparation.