Studies on the growth ofThiobacillus ferrooxidans

Summary Uranyl sulphate (0.2–0.9 mM) inhibited ferrous iron oxidation by growing cultures ofThiobacillus ferrooxidans. The addition of 5–100 mM uranium to the cultures caused immediate cessation of carbon dioxide fixation, rapid loss of viability and gradual depression of ferrous iron oxidation. Virtually no uranium was found in washed cells grown in the presence of subtoxic to toxic amounts of uranyl sulphate. Uranium-poisoned organisms appeared plasmolyzed in electron micrographs. Cultures tolerant to 5 mM UO₂ ²⁺ were develoepd by successive subculturing in increased uranium concentrations. The tolerance was maintained during subculturing in uranium-free medium. Frequency of mutants resistant to 1.0 and 1.5 mM UO₂ ²⁺ was 1 per 1.3×10⁶ and 1 per 9.0×10⁸, respectively. The frequency was increased in the presence of 15–150 mM nickel, zinc and manganese. In liquid cultures, bivalent cations and EDTA alleviated the toxicity of 2 mM uranyl sulphate.     

Comparison of 5′-Nucleotidase Activities Among Cultured Murine Melanoma Cell Lines

The activity of 5′-nucleotidase and ouabain-sensitive Na/K ATPase was determined in seven different mouse melanoma cell lines. Ouabain-sensitive Na/K ATPase activity was found in NP40-treated cell homogenates of all cell lines. However, 5′-nucleotidase activity was found in only one mouse melanoma cell line—JB/RH. The absence of expression of 5′-nucleotidase activity in the other six cell lines is not associated with pigmentation in melanoma cells, nor is the gene switched off in all transformed melanocytes of C57BL/6 origin.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Synthesis of polypeptides by microwave heating II. Function of polypeptides synthesized during repeated hydration-dehydration cycles

Summary Polypeptides, synthesized from a mixture of amino acid amides by microwave heating during repeated hydration-dehydration cycles, showed hydrolase- and oxidoreductase-like catalytic activities. Poly(GAVDH), polypeptides synthesized from an equimolar mixture (each 0.1 M) of glycinamide,l-alaninamide,l-valinamide,l-aspartic acid -amide, andl-histidinamide, catalyzed the hydrolysis of PNPAc. The hydrolytic rate of PNPAc with poly(GAVDH) was the quadruple of that ofl-histidine alone. Though the k_cat values of different resulting polypeptides were 10³–10⁶ times less than those of native hydrolases, the K_m value of the polypeptides further containing phenylalanine residues was nearly equal to that of the esterase. This result indicates the presence of hydrophobic interaction between a substrate and the polypeptides. Resulting polypeptides also showed catalytic activity for the reduction of ferricyanide ion [Fe(CN)^3–] with NADH. The polypeptides seemed to have a strong affinity for adenine nucleotides, because the reaction was inhibited by adenine derivatives such as NAD⁺ and AppA. A hypothesis for the emergence of primitive protein enzymes is discussed.     

Presence of calmodulin-like calcium-binding protein in Bacillus cereus T spores

Abstract Bacillus cereus T spores were extensively washed, broken, and heated at 90°C for 2 min. Using calcium-dependent hydrophobic interaction chromatography, a single peak protein fraction was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein fraction was retained by hydrophobic matrices (Phenyl-Sepharose) or a calmodulin antagonist (naphthalenesulfonamide) in a calcium-dependent manner. Calcium binding ability was verified by ⁴⁵Ca autoradiography and a competitive calcium binding assay using Chelex-100. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction was approx. 200-fold greater than the crude spore extract. Thus, B. cereus T spores have a calcium-binding protein with calmodulin-like properties.     

Iron-regulated membrane proteins and bacterial virulence

The amount of iron that might be readily available to bacteria in body fluids is extremely small. This iron restricted environment induces phenotypic changes in the metabolism and in the composition of the membrane proteins of bacteria growingin vivo. These changes are now providing a fresh insight into the capabilities of bacteria to multiply in host tissues and are suggesting new possibilities for targetting therapeutic and prophylactic measures.     

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.     

Causal connection between detoxification enzyme activity and consumption of a toxic plant compound

Insect herbivores can increase their detoxification activities against a particular plant poison in response to prolonged ingestion of the same compound. For example, larval tobacco hornworms (Manduca sexta) experience a dramatic increase in cytochrome P450 activity against nicotine after ingesting nicotine. While it is generally assumed that this induction process permits increased consumption of toxic plant tissues, we are not aware of any direct experimental support for this assumption. Using a two-tiered approach, we examined the functional significance of P450 induction to M. sexta larvae ingesting a toxic but non-deterrent concentration of nicotine. First, we related the time-course of P450 induction in midgut microsomes to changes in nicotine consumption. When offered a nicotine diet, larvae failed to show a significant increase in consumption before 36 h, which was coincident with the time-course of the induction of midgut P450 activities against aldrin and nicotine. Second, we determined whether inhibiting the induced P450 activities affected nicotine consumption. We found that the increase in nicotine consumption following the induction of nicotine metabolism could be strongly inhibited by treatment with piperonyl butoxide, which by itself did not inhibit consumption. These results provide direct evidence for a causal connection between P450-mediated detoxification activity and consumption of a toxic plant compound.Abbreviation PB piperonyl butoxide     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.