Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.     

Cystathionine β-synthase from human liver: Improved purification scheme and additional characterization of the enzyme in crude and pure form

The previously published procedure (Kraus et al. (1978) J. Biol. Chem.253, 6523–6528) for the purification of cystathionine β-synthase [l-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5′-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate “aged” for 7 days at 4 °C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. “Aging” of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine β-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystationine β-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme.     

Isolation of uroporphyrin III from Clostridium thermoaceticum

$\text{Clostridium thermoaceticum}$ contains a porphyrin which, based on visible absorption and ¹H nmr spectra as well as on chromatographic behavior, has been identified as uroporphyrin III.     

The activation of phospholipase C from Clostridium Welchii by quinine: an absolute requirement for calcium ions.

Quinine activates the hydrolysis of phosphatidyl choline suspensions by phospholipase C (E.C. 3.1.4.3) obtained from Clostridium welchii. Low levels of calcium are an absolute requirement for this activation: Mg2+, Ba2+, Sr2+, and Zn2+ are ineffective. The induction period, or lag phase for this enzyme is dependent upon both calcium concentration and substrate interfacial surface area. At low concentrations (less then 50 muM) calcium ions affect the induction period but not the maximal rate of hydrolysis, whereas guinine predominantly affects the rate of hydrolysis by alterations in the surface charge carried by the substrate.     

Dihydroorotase from Clostridium oroticum is an allosteric enzyme.

Dihydroorotase from $\text{Clostridium}$$\text{oroticum}$ exhibits allosteric behavior with respect to both of its substrates. L-dihydroorotate dependence reflects a positive homotropic interaction for which the Hill coefficient is 1.3–1.6, depending upon the preparation. Conversely, a negative homotropic response is observed when L-ureidosuccinate serves as substrate, as characterized by a Hill coefficient of 0.65–0.75. Interaction between L-dihydroorotate binding sites is a labile characteristic lost during enzyme purification. Negative cooperativity of ureidosuccinate binding appears to be more stable. The effects of purification and medium are also discussed.     

Semliki Forest virus membrane proteins, preparation and characterization of spike complexes soluble in detergent-free medium

After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95 · 10⁴ and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as “rosette”-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

A new sialyloligosaccharide from human milk: isolation and characterization using anti-oligosaccharide antibodies

A previously undescribed sialyloligosaccharide has been isolated from human milk using a specific anti-sialyloligosaccharide antibody. Structural studies of the radiolabeled oligosaccharide by enzyme degradation and binding by specific anti-oligosaccharide sera are consistent with the following structure: (sequence in text) The oligosaccharide is present only in milk from donors who secrete A, B, or H blood group substances; this is consistent with the requirement of at least one copy of the Se (Secretor) gene necessary for the synthesis of oligosaccharides with Fuc alpha 1-2Gal . . . linkages.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: comparison with the bacterial collagenase from Achromobacter iophagus.

The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl-l-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds $\text{Leu}\text{15}\text{-}\text{Tyr}\text{16}\text{and}\text{Leu}\text{11}\text{Val}\text{12}$ as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Isolation and characterization of liver glycogen synthase from diabetic rats

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M_r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M_r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate pI values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.     

Purification and characterization of glycolate oxidase from pumpkin cotyledons

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

A radioimmunoassay for human glucose phosphate isomerase: Measurement of immunoreactive enzyme from genetic variants

Glucose phosphate isomerase has been isolated to homogeneity from human placenta and monospecific antibody generated against the protein. The enzyme was radiolabeled with ¹²⁵I and purified by specific substrate-induced elution from cellulose phosphate. A radio-immunoassay was developed and the ratio of catalytic activity to immunoreactivity of the enzyme (molecular specific activity) was determined in hemolysates from five individuals heterozygous for genetic variant forms of the enzyme. In four cases involving point mutations no change in the molecular specific activity was noted. However, in a fifth case involving a deletion mutation, a decreased molecular specific activity was observed. Erythrocytes were fractionated on a density gradient and the molecular specific activity of the enzyme was measured in cells of different ages. A lower molecular specific activity was found in older cells, suggesting the accumulation of catalytically inactive, immunoreactive material during the aging of the erythrocyte.     

Isolation and characterization of a translation inhibitor from human term placenta

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.     

Dictyostelium calmodulin: affinity isolation and characterization

The Ca2+-binding regulatory protein calmodulin (CaM) has been purified from the cellular slime mold, Dictyostelium discoideum. Isolation of homogeneous Dictyostelium CaM was accomplished in high yield by ion-exchange chromatography and Ca2+-dependent affinity chromatography on phenothiazine-Sepharose 4B. This isolate has been demonstrated to possess the following physicochemical and functional properties characteristic of other CaM isolates: (i) a molecular weight ca. 16,000; (ii) an amino acid composition similar to other CaMs--with the notable exception that Dictyostelium CaM, as first determined by Bazari and Clarke [(1981) J. Biol. Chem. 256, 3598-3603] lacks the single trimethylated lysine (Tml) residue identified in nearly all CaMs purified to date; (iii) a CNBr peptide map similar to that of other CaMs; (iv) a Ca2+-dependent shift in migration during native- and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses; (v) ability to form Ca2+-dependent complexes with rabbit skeletal muscle troponin I; and (vi) ability to activate in a Ca2+-dependent manner bovine brain cyclic nucleotide phosphodiesterase.