Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

Purification and metal requirements of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH₄)₂SO₄ fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

UDP-glucose-4-epimerase from Poterioochromonas malhamensis

UDP-glucose-4-epimerase of Poterioochromonas malhamensis, Peterfi has been purified to apparent electrophoretic homogeneity. The enzyme has an apparent MW of 120 000 as determined by gel filtration of the active enzyme. Sodium dodecylsulfate polyacrylamide gel electrophoresis gave a MW of 59 000, thus indicating a dimeric structure. The epimerase does not require external NAD for activity. The apparent K_m values for UDP-glucose and UDP-galactose were calculated to be 1.67 mM and 0.26 mM, respectively. The pH optimum is at pH 8.7 and the isoelectric point is at pH 5.1 ± 0.15.     

Urease of Spirulina maxima

Urease (EC 3.5.1.5) was purified from Spirulina maxima by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200. The enzyme had maximum activity at pH 8.7, a K_m for urea of 0.12 mM and a MW of ca 232 000. A MW of 38 000 was determined for the subunits. The enzyme was inactivated by p-hydroxymercuribenzoate.     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.     

Characterization of wheat o-diphenolase isoenzyme

A highly purified isoenzyme of wheat o-diphenolase was characterized. The isoenzyme had a MW of ca 115 000, as determined by Sephadex G-100 gel filtration. The copper content was 0.20%, and the amino acid composition was determined. Two subunits (MWs ca 30 000 and 23 500) were detected by SDS gel electrophoresis. The K_m was found to be 5.1 mM for 4-methylcatechol and kinetic analysis showed that the isoenzyme exhibited substrate inhibition. The isoenzyme was characterized by its response to some inhibitors.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

Purification and Characterization of Two Epoxide Hydrolases from Corynebacterium sp. Strain N-1074

Enzymes II_a and II_b, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from Corynebacterium sp. strain N-1074, which catalyzes the formation of (R)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II_a for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II_b. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.     

Trichinella spiralis: Proteins and antigens isolated from a large-particle fraction derived from the muscle larva

Proteins and antigens derived from a large-particle fraction of muscle larvae of Trichinella spiralis (i.e., the S₃ fraction) were characterized in terms of their molecular weights, isoelectric points, carbohydrate contents, electrophoretic mobilities, antigenicity, and their ability to induce protection in mice. Gel filtration on Sephacryl S-200 yielded 5 major peaks of material while electrophoresis in polyacrylamide gel with sodium dodecyl sulfate revealed a minimum of 28 proteins ranging in MW from 11,000 to 200,000. Analytical isoelectric focusing on acrylamide gel yielded 37 bands of protein, while the periodic acid-Schiff reaction performed on a similar gel revealed 22 glycoproteins. Most proteins were within a pI range of 4.0–7.0, while all of the glycoproteins had pI ranging from 4.0 to 6.5. Immunoelectrophoresis of the S₃ fraction using hyperimmune rabbit serum demonstrated a minimum of 19 precipitin arcs, while crossed immunoelectrophoresis yielded 16 peaks. These determinations were made on several batches of material isolated in the same fashion and gave the same results. Preparative isoelectric focusing yielded 30 fractions. These fractions were assayed for the presence of antigens, then pooled and tested for their ability to induce protection in mice against an oral challenge infection. Fused rocket immunoelectrophoresis of all 30 fractions revealed the presence of a minimum of 18 antigens with pI ranging from 4.0 to 9.0. The pooled fractions (i.e., 1–9; 10–20; 21–30) all protected mice against oral challenge infection, while fraction 5 (pI = 4.3) protected best.     

Dihydrofolate reductase from soybean seedlings: Characterization of the enzyme purified by affinity chromatography

Dihydrofolate reductase from soybean seedlings has been purified by agarose-formylaminopterin affinity chromatography. The enzyme is homogeneous as judged by disc gel electrophoresis and immunodiffusion. Analysis by both Sephadex G-200 column chromatography and Sephadex (superfine) G-200 thin-layer gel filtration gives a molecular weight of about 140,000 for the enzyme. Sodium dodecyl sulfate-gel electrophoresis reveals the presence of nonidentical subunits. The enzyme contains nine sulfhydryl groups and is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 5,5-dithiobis(2-nitrobenzoic acid). Folate analogs methotrexate, aminopterin, and formylaminopterin cause potent inhibition of the enzyme, with I₅₀ values (concentration required for 50% inhibition) of 0.25, 0.63, and 1.78 μm respectively. The turnover number of the enzyme is 57. K_m values for dihydrofolate and NADPH are 35 and 415 μm, respectively. Dihydrofolate, but not NADPH, affords protection against heat inactivation and the protection constant, K_p (concentration of dihydrofolate at which half the original activity is retained), is 81 μm.     

Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.

The possibility that passage of tetracycline across the outer membrane of $\text{E.}\text{coli}$ K-12 is controlled by one or more of the proteins I_a, I_b and II¹ (Henning's nomenclature) was investigated. A mutant lacking protein I_a (obtained by selection for resistance to phage TuI_a) was more resistant to tetracycline than wild-type strains or those lacking only proteins I_b or II¹. The envelope protein composition of a tetracycline-resistant mutant ($\text{cmlB}$) was altered in several respects, but the major change involved loss of protein I_a. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein I_a plays a significant role in the passage of this antibiotic across the outer membrane.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Purification and characterization of L-mimosine synthase from Leucaena leucocephala

L-Mimosine synthase has been isolated from Leucaena leucocephala seedlings and purified 280-fold by heat treatment, ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. The enzyme was shown to be homogeneous by gel electrophoresis (MW 64 000±2000) and to consist of two identical subunits with MWs of 32 000±2000. The purified enzyme has a K_m value of 6.25 x 10^?3 M for O-acetyl-L-serine and 5.0 x 10^?3 M for 3,4-dihydroxypyridine. In these and other properties, the enzyme differs from β-(pyrazol-1-yl)-L-alanine synthase from Citrullus vulgaris seedlings.     

Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase

F_OF₁ ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F_OF₁. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b_I and b_II). The results show the b dimer to be located at a non-catalytic α/β cleft, with b_I close to subunit α, whereas b_II is proximal to subunit β. Furthermore, b_I can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-b_I-α, b_II-β, α-b_I-b_II-β, and a-b_I-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b_I is functionally related to the single b subunit present in mitochondrial ATP synthase.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.