Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Capture of latex beads, bacteria, endotoxin, and viruses by charge-modified filters.

This report demonstrates how electropositive filters can be used to enhance the removal of microorganisms and other negatively charged particles from water. It was shown that electropositive depth filters were capable of adsorbing viruses and endotoxins many times smaller than the average pore size of the filter. Electronegative filters of similar porosity or electropositive filters that had been treated to destroy the positive charge were almost ineffective under similar conditions for the removal of viruses and small latex spheres. The results of this study indicate that electropositive filters are highly effective in the removal of a wide range of contaminants over a wide range of pH values and ionic conditions.     

Characterization of dicyclohexylcarbodiimide binding sites in beef-heart mitochondria.

Binding studies with [¹⁴C]-dicyclohexylcarbodiimide showed the presence of binding sites in the beef-heart mitochondrial membrane at a concentration of 1.8 nmol/mg protein (1.4 sites per cytochrome a+a₃). Saturation of these sites correlated with the inhibition of the ATPase activity. The maximum binding capacity could be related with the amount of F₁-ATPase in mitochondria from various tissues.     

Continuous rearing of the aster leafhopper, Macrosteles fascifrons, on a chemically defined diet

A holidic diet for feeding the aster leafhopper, Macrosteles fascifrons, was formulated. The amino acids, B-vitamins, and sucrose are less concentrated than in aphid diets. Cholesterol, at 5 mg/ml, is required for the last ecdysis. Although leafhoppers reared on this diet have poorer survival and shorter life span than those reared on plants, they produce more progeny. Leafhoppers reared on this diet have completed the ninth generation and the culture is still thriving.     

The regulation of glycerol 3-phosphate oxidase of rat brownadipose tissue mitochondria by long-chain free fatty acids

Added free fatty acids inhibit oxidation of glycerol 3-phosphate, succinate and NADH in brown-adipose tissue mitochondria from 10-day-old rats. The most pronounced is the inhibitory effect of glycerol 3-phosphate cytochrome c reductase (GP-cyto. c reductase). Contrary to other reductases, GP-cyto. c reductase activity of freshly isolated mitochondria is already inhibited by the fraction of endogenous free fatty acids. Both added and endogenous free fatty acids inhibition of GP-cyto. c reductase is fully reversible by the removal of free fatty acids by bovine serum albumine treatment. The inhibition of GP-cyto. c reductase is of strictly non-competitive type. The most inhibitory are unsaturated long-chain free fatty acids-oleic and linoleic acid. Results are discussed with regards to the regulatory importance of free fatty acids in brown-adiposetissue during intensive non-shivering thermogenesis.     

Zoogeography of Intertidal Communities in the West Indian Ocean as Determined by Ocean Circulation Systems: Patterns from the Tetraclita Barnacles

The Indian Ocean is the least known ocean in the world with the biogeography of marine species in the West Indian Ocean (WIO) understudied. The hydrography of WIO is characterized by four distinct oceanographic systems and there were few glacial refugia formations in the WIO during the Pleistocene. We used the widely distributed intertidal barnacle Tetraclita to test the hypothesis that the distribution and connectivity of intertidal animals in the WIO are determined by the major oceanographic regime but less influenced by historical events such as Pleistocene glaciations. Tetraclita were studied from 32 locations in the WIO. The diversity and distribution of Tetraclita species in the Indian Ocean were examined based on morphological examination and sequence divergence of two mitochondrial genes (12S rDNA and COI) and one nuclear gene (histone 3, H3). Divergence in DNA sequences revealed the presence of seven evolutionarily significant units (ESUs) of Tetraclita in WIO, with most of them recognized as valid species. The distribution of these ESUs is closely tied to the major oceanographic circulation systems. T. rufotincta is distributed in the Monsoonal Gyre. T. ehsani is present in the Gulf of Oman and NW India. Tetraclita sp. nov. is associated with the Hydrochemical Front at 10°S latitude. T. reni is confined to southern Madagascan and Mauritian waters, influenced by the West Wind Drift. The endemic T. achituvi is restricted to the Red Sea. Tetraclita serrata consists of two ESUs (based on mtDNA analysis) along the east to west coast of South Africa. The two ESUs could not be distinguished from morphological analysis and nuclear H3 sequences. Our results support that intertidal species in the West Indian Ocean are associated with each of the major oceanographic circulation systems which determine gene flow. Geographical distribution is, however, less influenced by the geological history of the region.     

New insights to the ubiquitin–proteasome pathway (UPP) mechanism during spermatogenesis

Spermatogenesis is a complicated and highly ordered process which begins with the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. After meiosis, several morphological changes occur during spermatogenesis. During spermatogenesis, many proteins and organelles are degraded, and the ubiquitin–proteasome pathway (UPP) plays a key role in the process which facilitates the formation of condensed sperm. UPP contains various indispensable components: ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase enzyme E3 and proteasomes. At some key stages of spermatogenesis, such as meiosis, acrosome biogenesis, and spermatozoa maturation, the ubiquitin-related components (including deubiquitination enzymes) exert positive and active functions. Generally speaking, deficient UPP will block spermatogenesis which may induce infertility at various degrees. Although ubiquitination during spermatogenesis has been widely investigated, further detailed aspects such as the mechanism of ubiquitination during the formation of midpiece and acrosome morphogenesis still remains unknown. The present review will overview current progress on ubiquitination during spermatogenesis, and will provide some suggestions for future studies on the functions of UPP components during spermatogenesis.