Asbestos-catalysed lipid peroxidation and its inhibition by desferroxamine.

In an effort to understand the properties of asbestos fibres that might contribute to their being toxic, we incubated three different varieties of asbestos with phospholipid emulsions and looked for evidence of lipid peroxidation. Although all three types of asbestos were able to catalyse lipid peroxidation in the native state, this catalytic activity was inhibited by pre-washing of the asbestos with the iron chelator desferroxamine. This suggests that: lipid peroxidation may be one of the mechanisms by which asbestos produces tissue injury, and treatment with iron chelators might diminish the potential to produce this injury.     

Pectic Acid Lyases of Bacillus polymyxa

Four enzymes were separated from an extracellular preparation of Bacillus polymyxa by carboxymethylcellulose column chromatography. The pH optima were 8.3 to 8.5, 8.7 to 8.9, 9.2 to 9.4, and 9.5 to 9.6. All of the enzymes required calcium ion for maximum activity, whereas strontium ion was only partially effective in stimulating activity. Cobalt was the only other cation tested which was effective in two of the enzymes. The lyases seem to attack a calcium salt-bridged substrate. K(m) and V(m) data of the four enzymes on various oligomers are presented as well as paper chromatographic evidence of preferred sites of attack. All of the enzymes are endo-enzymes which, based upon their characteristics, were classed into two types.     

The endocytosis of asbestos by mouse peritoneal macrophages and its long-term effect on iron accumulation and labyrinth formation

The effect of a single intraperitoneal injection of crocidolite asbestos fibres on the peritoneal cell population were studied. Attention was paid to the changes in the proportions taken by the various types of cell in this population after peritoneal stimulation as well as the handling of asbestos fibres by the peritoneal cells and the formation of asbestos bodies. Intraperitoneal administration of crocidolite led to an influx of inflammatory cells into the peritoneal cavity. The asbestos fibres were phagocytosed and gradually cleared from the peritoneal cavity. Long before this clearance was completed, the peritoneal cell population had returned to the steady state. The stimulated peritoneal macrophages showed increasing concentrations of iron in both lysosomes and the cytoplasm. At later time points, residual bodies containing iron and asbestos fibres were seen frequently in macrophages, but asbestos bodies were not found. As a reaction to the administration of crocidolite asbestos, macrophages from the peritoneal cavity develop tubular systems (labyrinths) that increase in number and size.     

Sulfotransferases, sulfatases and formylglycine-generating enzymes: a sulfation fascination

Sulfotransferases and sulfatases are the major enzymes responsible for sulfate transfer processes. The past two years have seen the elucidation of new functions for these enzymes, and a great progression in their structural characterization, which confirms that these two types of enzymes possess a highly conserved fold. For catalytic activity, sulfatases must contain a formylglycine residue, which is generated by various formylglycine-generating enzymes. Mechanistic and structural details have recently been obtained for a group of cofactor-independent formylglycine-generating enzymes termed FGEs. Finally, an increasing light has been cast upon the mechanism of sulfatase inactivation by a group of clinically important agents, the aryl sulfamates.     

The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

Enkephalinase: Selective inhibitors and partial characterization

There are at least two types of enzymes in brain, endopeptidases and aminopeptidases, which metabolize enkephalins. Evidence is presented to suggest that enkephalinase, an endopeptidase cleaving at the Gly-Phe bond, is specific for the endogenous enkephalinergic system. Selective inhibitors are described for each enzyme. These are parachloromercuriphenylsulfonic acid and puromycin in the case of aminopeptidases and various enkephalin fragments in the case of enkephalinase. Some characteristics of the two types of enzymes are described. Enkephalinase has many properties in common with the well-characterized brain angiotensin-converting enzyme. These two enzymes, however, behaved differently when tested for chloride dependance, for activity in several buffers and for susceptibility to specific inhibitors.     

Mutagenesis of Escherichia coli (CSH50) by asbestos

The mutagenic character of richterite asbestos was detected in Escherichia coli strain CSH50, using a modified Ames test. Two sets of experimental plates which contained naturally occurring asbestos were used, to one set of which S-9 rat liver homogenate was added. Control plates contained no asbestos. Mutant colonies appeared in significantly greater frequency in both experimental sets as compared with controls, and experimental plates with rat liver homogenate contained the greatest number of mutant colonies. It is postulated that enzymes contained in rat liver metabolize some unknown mutagen introduced with the natural asbestos sample other than the inert richterite fibers themselves. This finding, together with the unlikelihood that bacterial cell walls could be easily penetrated by asbestos fibers, tends to negate fiber penetration as the mechanism of mutagenesis. The mutagenic mechanism operative in E. coli (CSH50) may be applicable to animal cell systems.     

Certification of Probability of Sterilization of Liquid by Filtration

Four types of hydrosol filters, two reusable (diatomaceous cylinder and fritted-glass funnel) and two disposable (asbestos pad and membrane filter) were challenged with a heavy bacterial suspension to assess their ability to produce sterile filtrates. Two of the four diatomaceous earth filters, the four fritted-glass funnels, and all of the asbestos pads tested generally gave sterile filtrates. However, only one type of filter, one of the membranes in its manufacturer's own holder, consistently gave sterile filtrates. The two other types of membranes usually gave sterile filtrates if tested in one manufacturer's holder, but all types invariably gave contaminated filtrates when tested in another manufacturer's holder. Contaminated filtrates were generally attributed to a poor reusable filter or to a faulty holder used with a disposable filter. If a high degree of certainty is required for sterile heat-labile filtrate, it is suggested that the liquid be passed through two or more filters in a previously tested and proven system.     

N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.     

Purification of Penicillin G Amidase using Quaternary Ammonium Salts and effect on the activity of the immobilised enzymes

This paper describes the purification of Penicillin G Amidase (EC 3.5.1.11) using quaternary ammonium salts with the aim of increasing the activity of immobilised enzymes prepared from the purified solutions. Two different quaternary ammonium salts were tested with different solutions of the enzyme. It was concluded that the quaternary ammonium salts used selectively precipitated the non-enzymatic protein leaving in solution practically all the enzyme resulting in a high yield of purification. Optimal conditions for purification using the two types of quaternary ammonium salts were determined. Immobilisation studies were performed from various purified enzyme solutions, using different amounts of a quaternary ammonium salt. The immobilised enzymes so obtained showed a much higher activity than the immobilised enzyme obtained from non-purified enzyme solutions.     

The effect of the presence of globular proteins and elongated polymers on enzyme activity

We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.     

The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes

In the plasma membranes from several mammalian tissues (including normal and tumor tissues), a Mg2+ (or Ca2+)-dependent ATP phosphohydrolase activity is present in much greater amount than the (Na+ + K+)-ATPase. The ouabain-insensitive activity can be attributed to at least two enzymes, an ATPase (EC 3.6.1.3) and an ATP diphosphohydrolase (EC 3.6.1.5). The ATPase hydrolyzes ATP and other nucleoside triphosphates and is not inhibited by azide. The ATP diphosphohydrolase hydrolyzes both ATP and ADP (and other nucleoside tri- and diphosphates) and the hydrolysis of adenine nucleotides is strongly inhibited by 10 mM azide. The ratios of these two enzymes in the various membranes (as determined by the extent of azide inhibition) vary widely. The ATP diphosphohydrolase accounts for most of the Mg2+ (or Ca2+)-dependent ATP hydrolysis activity of the plasma membranes of liver (mouse), kidney (dog), two mouse sarcomas, and a human astrocytoma (xenograft in athymic mice). The ATPase is more dominant in the plasma membranes from mouse brain and human oat cell carcinoma. The widespread presence of the ATP diphosphohydrolase in plasma membrane from various types of tissues is demonstrated for the first time and is of particular interest in view of its relatively high activity in the plasma membranes of two sarcomas. The membrane-bound ATP diphosphohydrolase is characterized with respect to its metal ion activators, substrates, and inhibitors. These results should facilitate the distinction of this enzyme from other ATP hydrolyzing enzymes of plasma membranes in future investigations.     

Comparison of glycogen phosphorylase kinases of various rat tissues

Glycogen phosphorylase kinases in soluble fractions of various rat tissues were examined for the pH 6.8/8.5 activity ratio, Ca2+-dependency, activation by cyclic AMP-dependent protein kinase (protein kinase A), and reactivity with anti-skeletal muscle phosphorylase kinase serum. The enzymes could be divided into at least two major groups; muscle and liver types. The muscle type, that has a low value of pH 6.8/8.5 activity ratio, is highly dependent on Ca2+, markedly activated by protein kinase A, and strongly inhibited by the antiserum. Inversely, the liver type, that has a high value of pH 6.8/8.5 activity ratio, is poorly dependent on Ca2+, not activated by protein kinase A, and weakly inhibited by the antiserum. The enzymes from heart and skeletal muscle were similar and belonged to the former entity. Whereas, the enzymes from liver, kidney, spleen, lung, and testis appeared to belong to the latter entity. The enzyme from brain apparently differs from these entities, and seems to be an intermediate type or a hybrid of the two.     

Asbestos-induced pulmonary toxicity: role of DNA damage and apoptosis

Asbestos causes asbestosis and various malignancies by mechanisms that are not clearly defined. Here, we review the accumulating evidence showing that asbestos is directly genotoxic by inducing DNA strand breaks (DNA-SB) and apoptosis in relevant lung target cells. Although the exact mechanisms by which asbestos causes DNA damage and apoptosis are not firmly established, some of the implicated mechanisms include the generation of iron-derived reactive oxygen species (ROS) as well as reactive nitrogen species (RNS), alteration in the mitochondrial function, and activation of the death receptor pathway. We focus on the accumulating evidence implicating ROS. DNA repair mechanisms have a key role in limiting the extent of DNA damage. Recent studies show that asbestos activates DNA repair enzymes such as apurinic/apyrimidinic endonuclease (APE) and poly (ADP-ribose) polymerase (PARP). Asbestos-induced neoplastic transformation may result in the setting where DNA damage overwhelms DNA repair in the face of a persistent proliferative signal. Strategies aimed at limiting asbestos-induced oxidative stress may reduce DNA damage and, as such, prevent malignant transformation.     

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.     

The biomedical and epidemiological characteristics of asbestos-related diseases: a review

The purpose of this paper is to provide the reader with an overview of the biomedical and epidemiological characteristics of asbestos-related disease based upon currently available information. Epidemiological and experimental data developed over the past 20 years have greatly added to our knowledge of the biological effects of asbestos, particularly in relation to clinical disease. This information has substantially strengthened the evidence linking asbestos to specific health effects. Lung cancer and mesothelioma are clearly the most important asbestos-related causes of death among exposed individuals, although the accumulated data is suggestive of the existence of an excess risk of gastrointestinal and a variety of other neoplasms. Animal studies confirm the human epidemiological results and indicate that all commercially available fiber types are capable of producing lung cancer and mesothelioma. Experimental implantation and injection studies also show that the carcinogenicity of mineral fibers (including asbestos) is directly related to their dimensionality and not their chemical composition. Although the asbestos-related medical and scientific literature is voluminous, many issues related to the biological activity of asbestos fibers are as yet unresolved. Due to experimental and analytical limitations, questions concerning risk at low-level exposure, dose-response relationships, and individual susceptibility remain problematic.     

The molecular basis of two contrasting metabolic mechanisms of insecticide resistance

The esterase-based insecticide resistance mechanisms characterised to date predominantly involve elevation of activity through gene amplification allowing increased levels of insecticide sequestration, or point mutations within the esterase structural genes which change their substrate specificity. The amplified esterases are subject to various types of gene regulation in different insect species. In contrast, elevation of glutathione S-transferase activity involves upregulation of multiple enzymes belonging to one or more glutathione S-transferase classes or more rarely upregulation of a single enzyme. There is no evidence of insecticide resistance associated with gene amplification in this enzyme class. The biochemical and molecular basis of these two metabolically-based insecticide resistance mechanisms is reviewed.     

Enzymes of nucleotide metabolism: the significance of subunit size and polymer size for biological function and regulatory properties

The 72 enzymes in nucleotide metabolism, from all sources, have a distribution of subunit sizes similar to those from other surveys: an average subunit Mr of 47,900, and a median size of 33,300. The same enzyme, from whatever source, usually has the same subunit size (there are exceptions); enzymes having a similar activity (e.g., kinases, deaminases) usually have a similar subunit size. Most simple enzymes in all EC classes (except class 6, ligases/synthetases) have subunit sizes of less than 30,000. Since structural domains defined in proteins tend to be in the Mr range of 5,000 to 30,000, it may be that most simple enzymes are formed as single domains. Multifunctional proteins and ligases have subunits generally much larger than Mr 40,000. Analyses of several well-characterized ligases suggest that they also have two or more distinct catalytic sites, and that ligases therefore are also multifunctional proteins, containing two or more domains. Cooperative kinetics and evidence for allosteric regulation are much more frequently associated with larger enzymes: such complex functions are associated with only 19% of enzymes having a subunit Mr less than or equal to 29,000, and with 86% of all enzymes having a subunit Mr greater than 50,000. In general, larger enzymes have more functions. Only 20% of these enzymes appear to be monomers; the rest are homopolymers and rarely are they heteropolymers. Evidence for the reversible dissociation of homopolymers has been found for 15% of the enzymes. Such changes in quaternary structure are usually mediated by appropriate physiological effectors, and this may serve as a mechanism for their regulation between active and less active forms. There is considerable structural organization of the various pathways: 19 enzymes are found in various multifunctional proteins, and 13 enzymes are found in different types of multienzyme complexes.     

Aromatic Biosynthesis in Higher Plants: IV. THE DISTRIBUTION OF DEHYDROSHIKIMIC REDUCTASE AND DEHYDROQUINASE

The distribution of two of the enzymes involved in the biosynthesisof aromatic compounds in higher plants, namely dehydroshikimicreductase and dehydroquinase, was studied. The two enzymes werefound to occur mainly in the supernatant fraction of cauliflowerbuds. In extracts of pea epicotyls, 30 per cent. of the dehydroshikimicreductase activity was associated with the mitochondria. Theactivities of the two enzymes in various organs of germinatingpea seedlings were measured over an 8-day period. Synthesisof both enzymes was indicated in the roots, shoots, and cotyledons.The two enzymes were found to occur in a variety of plant materials.     

Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin.