Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca²⁺, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca²⁺ also affected substrate binding, as evidenced by a decrease in K_m for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Mechanism of Chloramphenicol Resistance in Staphylococcus epidermidis

The mechanism of chloramphenicol resistance in several multiple-resistant Staphylococcus epidermidis strains has been studied and shown to be due to the presence of the enzyme, chloramphenicol acetyltransferase. As with S. aureus, the inactivating enzyme in S. epidermidis appears to be the product of a structural gene on the chloramphenicol plasmid because resistance and enzyme activity are concurcurrently lost after growth in acridine orange or at elevated temperatures. The synthesis of chloramphenicol acetyltransferase in S. epidermidis has been compared with the function of a similar enzyme in chloramphenicol-resistant S. aureus with the conclusion that the kinetics of induction, products of the reaction, and general properties of the enzymes are identical. The chloramphenicol acetylating enzyme from S. epidermidis has been purified to a state of homogeneity and compared with the analogous purified S. aureus enzyme. Both purified preparations consist of native enzymes with molecular weights of 80,000, and evidence is presented that is consistent with their being made up of four identical subunits of 20,000 each. The two staphylococcal enzymes are identical with respect to pH optimum, apparent affinity (K_m) for chloramphenicol, heat denaturation, and immunological reactivity, but they differ in electrophoretic mobility, chromatographic behavior, substrate specificity, and sensitivity to inhibition by mercuric ion.     

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b₅, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Purification of a debranching enzyme (R-enzyme) from malted barley,and the role of the enzyme in the digestion of starch granules during the germination of barley seeds

A debranching enzyme (R-enzyme or pullulan-6-glucanohydrolase, EC3.2.1.41), free from contaminating carbohydrases and homogeneous by poly(acrylamide) disc-gel electrophoresis, has been purified from malted barley. A partially purified preparation of this enzyme (3.1 units/mg of protein) accelerated the rate of digestion of barley-starch granules by the action of purified alpha and beta amylases to the same extent as was effected by the dialyzed, crude extract from malted barley. Contrary to expectation, the debranching enzyme, purified to homogeneity (10 units/mg of protein), had very little accelerating effect. These results indicate that a factor or factors, which may be maltase or α-d-glucosidase and were lost during the purification of the debranching enzyme, may play a role in the digestion of starch granules by the dialyzed, crude extract from malted barley in vitro and by enzymes in the endosperm of germinating barley seeds in vivo. The debranching enzymes, including barley-malt R-enzyme, Aerobacter pullulanase, and Pseudomonas isoamylase, did not digest starch granules to a detecble extent.     

DNA-dependent RNA polymerases isolated from yeast mitochondria

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di-t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.     

Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C₄ enzyme to inhibition by malate is also unaffected by preincubation with these ligands.     

Enzyme therapy: Differential in vivo retention of bovine hepatic,renal, and splenic β-glucuronidases and evidence for enzyme stabilization by intermolecular exchange

The in vivo fates of β-glucuronidase partially purified from bovine hepatic, renal, and splenic tissues were compared. Following administration to β-glucuronidase-deficient mice, hepatic and renal enzymes were primarily recovered in murine liver lysosomes (70% of dose by 30 min) with a subsequent decline to undetectable levels by 24 h. However, recovery of the splenic activity was markedly prolonged; 25% of dose was recovered in murine liver at 24 h with a decline to undetectable levels at 68 h, which was three times longer than that observed for the hepatic or renal enzymes. When administered entrapped in autologous erythrocytes, prolonged retention was again observed only with splenic enzyme, suggesting that recognition processes were not responsible for the splenic retention. No physical or kinetic differences were observed when these enzymes were further purified and characterized by gel filtration, ion-exchange, and polyacrylamide or cellulose acetate electrophoreses. Mixing of the splenic with hepatic or renal enzymes prior to administration resulted in an apparent enhanced retention of the hepatic and renal enzymes, suggesting a unique subunit structure of the splenic isozyme which might enhance the in vivo stability of the hepatic or renal enzymes by subunit exchange. These results indicate that subtle differences in the enzymes derived from multiple tissue sources may be recognized in vivo and that tissue source should be a consideration for enzyme therapy.     

Non-Complexed Four Cascade Enzyme Mixture: Simple Purification and Synergetic Co-stabilization

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10⁹ mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.     

Thermal stability and redox properties of M. tuberculosis CuSOD

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker.     

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.     

Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from Brevibacterium helvolum

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of α-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.     

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used ³¹P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.