Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity

A comparison has been made between the 3,5-dinitrosalicylic acid (DNS) and alkaline copper methods of assaying for reducing sugars released during the enzymatic hydrolysis of cellulose by culture filtrates from Trichoderma harzianum E58. The DNS method was shown to be more readily influenced by the incubation conditions and by components derived from lignocellulosic substrates. The endo-1,4-β-d-glucanase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] values obtained with the DNS assay were always considerably higher than those obtained with the alkaline copper method and did not give reducing values that were proportional to the actual number of hemiacetal reducing groups. The alkaline copper assay was not affected by the degree of polymerization of the substrate. Although this latter method appeared to be superior to the DNS assay it was still affected by the incubation conditions, nature of the substrate and the influence of other cellulase components on each of the specific enzyme assays.     

The use of derivatives of 2-naphthol as substrates for the demonstration of carboxyl esterases in situ and the enhancement of the reaction with DMSO

Summary The selective localization of carboxyl esterase in broad bean root tip obtained with naphthyl AS acetate as against 1-naphthyl acetate is not due to the fact, that the mentioned substrate is a derivative of 2-naphthol, since with 2-naphthyl acetate, 6-benzoyl-2-naphthyl acetate and 6-brom-2-naphthyl acetate the results are—finally—the same as with 1-naphthyl acetate. The precipitation in the incubation media of some of these substrates was overcome by the addition of some solvents, DMSO being the most suitable in this respect. The remarkable shortening of the incubation time in the presence of DMSO is due to the enhanced penetration of the substrates to the reacting sites, since the substrate concentration is not critical under the given conditions. This is in favour of the presumptive localization of the enzyme revealed within membrane—bound particles of apparently lysosome—like nature.     

Enzyme studies on TPPase-reactive cytoplasmic structures observed in early meiotic prophase I of the hamster oocyte

Summary Ovaries of immature and adult hamsters were incubated in medium containing thiamine pyrophosphate (TPP) to determine the age at which TPPase-reactive cytoplasmic structures first appear in the germ cells, and at what age the structures cease to be present. The structures were found only in oocytes from animals 8–15 days of age. They occur in predictyate germ cells in polyovular follicles and in very early dictyate oocytes in unilaminar follicles. The TPPase-reactive structures were never observed in atretic oocytes, in unilaminar follicles of adult animals, nor in multilaminar follicles of animals at any age.Ovaries of 8–12-day-old animals and adults were then incubated in media in which one of the following substrates was substituted for TPP: uridine diphosphate (UDP), inosine diphosphate (IDP), and adenosine monophosphate (AMP). Half of the samples in each experiment were incubated in medium containing the inhibitor L-p-bromotet-ramisole. -Glycerophosphate was used in control incubations, or the substrate was omitted entirely.The cytoplasmic structures were found to be reactive after incubation in UDP-containing media, but not after incubation in media containing AMP. With IDP as substrate, reactions were atypical and confined to peripheral regions of the cytoplasm.Other sites of enzyme activity after incubation with the various substrates (cell membranes, zona pellucida, endoplasmic reticulum and Golgi apparatus) are also described and discussed.     

Probing the efficiency of proteolytic events by positional proteomics

Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.     

The caspase-like sites of proteasomes,their substrate specificity,new inhibitors and substrates,and allosteric interactions with the trypsin-like sites

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.     

Kinetic studies on glucoamylase of rabbit small intestine.

The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with alpha-1,4 and alpha-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.     

Comparison of populations of human faecal bacteria before and after in vitro incubation with plant cell wall substrates

Human faecal slurries were incubated anaerobically with larchwood xylan, oat spelt xylan, wheat bran, apple cell walls or sugar beet pulp as sole carbon sources. The populations which developed during incubation were different from the inoculum, the most marked changes being an increase in the number of Bacteroides species and a decrease in the number of Fusobacterium species for all carbon sources tested. With a water-soluble preparation of larchwood xylan the population was dominated by species able to ferment this substrate, in contrast to the population which developed with the insoluble substrates. The ability to use one plant cell wall substrate appeared to be related to the ability to use others. Strains capable of using plant cell wall substrates included Bacteroides spp., Clostridium clostridiiforme, Bifidobacterium longum, Fusobacterium spp. and Escherichia coli. When incubation with two contrasting substrates (bran and larchwood xylan) was replicated, the populations which developed were reproducibly different from the inoculum and from each other.     

Comparison of populations of human faecal bacteria before and after in vitro incubation with plant cell wall substrates

Human faecal slurries were incubated anaerobically with larchwood xylan, oat spelt xylan, wheat bran, apple cell walls or sugar beet pulp as sole carbon sources. The populations which developed during incubation were different from the inoculum, the most marked changes being an increase in the number of Bacteroides species and a decrease in the number of Fusobacterium species for all carbon sources tested. With a water-soluble preparation of larchwood xylan the population was dominated by species able to ferment this substrate, in contrast to the population which developed with the insoluble substrates. The ability to use one plant cell wall substrate appeared to be related to the ability to use others. Strains capable of using plant cell wall substrates included Bacteroides spp., Clostridium clostridiiforme, Bifidobacterium longum, Fusobacterium spp. and Escherichia coli. When incubation with two contrasting substrates (bran and larchwood xylan) was replicated, the populations which developed were reproducibly different from the inoculum and from each other.     

Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells. Introduction of T4 endonuclease V into frozen and thawed cells

Endonuclease V from E. coli infected with phage T4 was used to evaluate the frequency and the removal of pyrimidine dimers from DNA in cultured mammalian cells. Cellular membranes were made permeable to the enzyme by two cycles of rapid freezing and thawing. The number of endonuclease-sensitive sites in DNA was assayed by sedimentation in alkaline sucrose gradients upon which the cells were lysed directly. Comparison of the frequency of endonuclease-sensitive sites with the frequency of pyrimidine dimers determined by chromatographic analysis of hydrolysed DNA indicated that about 50% of the dimers in the permeabilized cells were substrates for T4 endonuclease V. This was confirmed by observation that when DNA treated with the enzyme in situ was purified, it contained the expected additional number of endonuclease-sensitive sites if again treated with the enzyme. The percentage of pyrimidine dimers recognized by T4 endonuclease V was enhanced to nearly 100% by exposing the permeabilized cells to 2 M NaCl before the enzyme was introduced. This method allowed the measurement of frequencies of endonuclease-sensitive sites after doses of UV irradiation at low as 0.5 J/m2. Loss of endonuclease sites from cellular DNA was observed during post-irradiation incubation of V79 Chinese hamster cells and several human cell strains. A comparison of the results obtained in human cells with or without the high-salt exposure before endonuclease treatment suggested that the dimers recognized under low-salt conditions may be removed slightly faster than those recognized only after high-salt exposure.     

Energy-dependent masking of substrate binding sites of the lactose permease of Escherichia coli

A method was devised to measure the number of specific substrate binding sites of lactose permease in membrane preparations derived from mechanically disrupted Escherichia coli.The method consists of incubation with radioactive thiodigalactoside (galactosyl β-d-thiogalactoside, TDG) followed by precipitation with 80% saturated (NH₄)₂SO₄ and washing with the same solution.The measurement gave reproducible results, easy to correct for a moderate nonspecific binding, but active transport, when it occurred, resulted in excess counts.The radioactivity bound to the pellet was shown to depend on the presence of intact lac y gene product.Addition of ascorbate and phenazine methosulfate (PMS) stimulated active transport into the membrane vesicles. This could be inhibited by cyanide and by uncoupling agents and under these conditions the number of available binding sites was strongly diminished, while the inhibitors alone did not bring about a similar decrease.The decrease of available substrate binding sites was reversed by removal of oxygen or by washing out the respiratory substrates.The decrease in available binding sites is interpreted as reflecting one of the energy coupling steps which during in vivo active transport prevents the mobile carrier from being available for outflux, but the detailed interpretation of the reported results raises a number of problems connected with the energy cycle of active transport     

Site-specific recombination of bacteriophage lambda. The change in topological linking number associated with exchange of DNA strands

The changes in supercoiling that accompany site-specific recombination have been measured. In each experiment, the substrate was a circle that contained two attachment sites oriented as an inverted repeat; recombination between the sites inverts one segment of the circle with respect to the other. Using conditions developed in the accompanying work, a measurable amount of the recombinant is in the form of unknotted, simple circles. The difference between the topological linking number of this product relative to that of the substrate can be determined directly from the change in mobility during agarose gel electrophoresis. With partially supercoiled substrates, both integrative and excisive recombination are characterized by a unique change in linking number, a relaxation of two topological turns. For excisive recombination, it has been possible to study closed circular substrates that lack supercoils. In this case, changes in linking number of both +2 and -2 are observed. These results are used to evaluate various proposals for synapsis and strand exchange in bacteriophage lambda site-specific recombination.     

Structural requirements of the phospholipid substrate for phospholipid N-methylation in rat liver

The role of endogenous phospholipid substrates for phospholipid methylation was investigated in rat liver microsomes. The amount of phosphatidylethanolamine could be drastically reduced by treatment of microsomes with an amino group-blocking compound, methylacetimidate. Simultaneously, the formation of labelled phospholipids from S-adenosyl[Me-3H]methionine decreased, indicating that the amount of endogenous substrate influenced the reaction rate. Phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and phosphatidylmonoethylethanolamine added as dispersions to untreated or treated microsomes stimulated phospholipid methylation, whereas several other phospholipids were inactive. In other experiments the role of phospholipid substrates in intact cells was studied. Cultured rat hepatocytes were enriched in different phospholipids by preincubation with different amino alcohols, and the effects of phospholipid methylation was measured by incubation with [Me-14C]methionine. Phospholipid methylation was significantly stimulated after preincubation with ethanolamine, monomethylethanolamine, monoethylethanolamine and 2-aminobutanol. The results show that both the number and chain length of N-alkyl substituents on phosphatidylethanolamine, as well as other changes in the ethanolamine moiety, will affect the ability of different phospholipids to act as methyl acceptors.     

The effect of substrate on the production of infectious virus by cells in culture

Herpes simplex virus type I (HSV-1), infectious bovine rhinotracheitis virus (IBR) and turkey herpesvirus were examined for growth in cells cultured on three different substrates. The substrates were glass, DEAE-dextran and collagen gel. With two of the viruses, HSV-1 and IBR, there were no apparent differences in production as a function of substrate. In contrast, the amount of the turkey herpesvirus which was recovered varied greatly with the substrate. Titers were highest on glass, followed by DEAE-dextran and then collagen gel. Our previous studies have indicated that the substrate on which anchorage-dependent cells are grown in vitro has an affect on a number of biological and biochemical properties. The present study indicates that the production of commercially important biologicals can be affected by the substrate.     

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites. Probing of modified DNA duplexes as activators of the enzyme.

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.     

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

Summary A soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with -d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.     

Amino acids as main substrates for sulfate-reducing bacteria in surface sediment of a eutrophic bay

The inner part of Tokyo Bay, Japan, is highly eutrophicated as shown by the frequent occurrence of red tide. The bottom water is anoxic during warm seasons especially at artificially dredged sites. In the sediment slurries prepared from surface sediment samples collected from the dredged sites, substrate addition stimulated the consumption of sulfate during anaerobic incubation. Of the substrates added, the seston composed mainly of diatom stimulated consumption more than lactate and acetate. Its effect was nearly equal to that of casamino acids. Casamino acids and some amino acids also accelerated the rate of sulfate reduction measured by the tracer method in sediment samples more than lactate or acetate. Anaerobic incubation of the sediment slurry amended with casamino acids showed that the consumption of amino acids was retarded by the addition of molybdate (final concentration; 20 mM). In the slurry amended with only molybdate, glutamate was accumulated distinctively and linearly with time. Its accumulation rate in molar base was comparable to the rate of sulfate reduction. These results suggested that amino acids were the main substrates for sulfate-reducing bacteria (SRB) in the sediment. The MPN values of SRB in these sediment samples were often higher with the enumeration medium containing casamino acids instead of lactate. Furthermore, during a week incubation of sediment slurries amended with substrates, casamino acids and seston more greatly stimulated the growth of SRB enumerated by both media than lactate.     

Alginate biosynthesis by Pseudomonas aeruginosa: effect of arsenite and other metabolic inhibitors

Biosynthesis of alginic acid in presence of metabolic inhibitors by resting cells of mucoid Pseudomonas aeruginosa was studied. Among the inhibitors tested, arsenite exhibited very interesting results, while the others showed no remarkable-effect. Firstly, arsenite stopped alginate production from all the substrates during initial hours of incubation; secondly, degradation of newly synthesized alginates to smaller molecular weight fragments took place if it was added after a few hours of incubation with the substrate; and thirdly, uncontrolled synthesis of alginate started after several hours of inhibition. Presence of arsenite was needed for the initial inhibitory phase of alginate synthesis; but once the cells were capable of synthesizing alginate after initial hours of inhibition, arsenite may be omitted from the medium.     

The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase

We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli. The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map. We cloned the gene in a high copy number vector and purified the enzyme. It is a monomer, with an apparent molecular weight of 65,000. The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose. We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates. The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin. In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose. To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation. More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose. Furthermore, [14C]maltotriose, specifically labeled at the reducing end, was hydrolyzed to [14C]glucose and unlabeled maltose. These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides. The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960. Homology to glucosidases, alpha-amylases, and pullulanases were observed. Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.     

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.     

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.