Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

Cloning and characterization of mouse klk27, a novel tissue kallikrein expressed in testicular Leydig cells and exhibiting chymotrypsin-like specificity.

A cDNA clone of a new mouse tissue kallikrein, designated mKlk27, was isolated from an adult mouse testis cDNA library. mKlk27 was expressed in the submaxillary glands and testis of the mouse. In testis, mKlk27 gene was expressed exclusively in the Leydig cells of the adult mouse. Active recombinant mKlk27 exhibited chymotrypsin-like cleavage specificity. A single amino-acid substitution of Gly for Asp at position 209 in mKlk27 resulted in complete loss of its chymotryptic activity but acquisition of tryptic activity. mKlk27 effectively hydrolyzed casein, gelatin and fibronectin. Insulin-like growth factor binding protein-3 was also hydrolyzed by recombinant mKlk27. These results suggest that mKlk27 plays an important role in association with the function of the adult mouse testis.     

Nucleotide binding sites and chemical modification of the chromaffin granule proton ATPase

The purified proton ATPase of chromaffin granules contains five different polypeptides denoted as subunits I to V in the order of decreasing molecular weights of 115,000, 72,000, 57,000, 39,000, and 17,000, respectively. The purified enzyme was reconstituted as a highly active proton pump, and the binding of N-ethylmaleimide and nucleotides to individual subunits was studied. N-Ethylmaleimide binds to subunits I, II, and IV, but inhibition of both ATPase and proton pumping activity correlated with binding to subunit II. In the presence of ADP, the saturation curve of ATP changed from hyperbolic to a sigmoid shape, suggesting that the proton ATPase is an allosteric enzyme. Upon illumination of the purified enzyme in the presence of micromolar concentrations of 8-azido-ATP, alpha-[35S]ATP, or alpha-[32P]ATP subunits I, II, and IV were labeled. However, at concentrations of alpha-[32P]ATP below 0.1 microM, subunit II was exclusively labeled in both the purified and reconstituted enzyme. This labeling was absolutely dependent on the presence of divalent cations, like Mg2+ and Mn2+, while Ca2+, Co2+, and Zn2+ had little or no effect. About 0.2 mM Mg2+ was required to saturate the reaction even in the presence of 50 nM alpha-[32P]ATP, suggesting a specific and separate Mg2+ binding site on the enzyme. Nitrate, sulfate, and thiocyanate at 100 mM or N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at 100 microM prevented the binding of the nucleotide to subunit II. The labeling of this subunit was effectively prevented by micromolar concentrations of three phosphonucleotides including those that cannot serve as substrate for the enzyme. It is concluded that a tightly bound ADP on subunit II is necessary for the activity of the enzyme.     

Internal anion binding site and membrane potential dominate the regulation of proton pumping by the chromaffin granule ATPase

Effects of anions and membrane potential on the reconstituted proton pump from chromaffin granules were investigated. When acetate was present inside of the vesicles, ATP-dependent proton uptake was absolutely dependent on external chloride. Without external chloride, however, substantial proton uptake was observed when chloride or sulfate was present inside of the vesicles. Inside negative membrane potential drove ATP-dependent proton uptake regardless of the anion species present inside or outside of the vesicles. It is concluded that the internal anion binding site and membrane potential regulate the proton pumping activity of the ATPase.     

Rhythmical jaw movements and lateral ponto-medullary reticular neurons in rats

Neuronal activity in the lateral reticular formation was investigated in urethane-anesthetized rats. Stimulation of anterior and posterior cortical areas induced two types of rhythmical jaw movements (RJM). The effects of stimulation of these cortical areas, the peripheral nerves, and the trigeminal motor nucleus on these neurons and their activity during the RJM were analyzed. The smallest percentage of neurons receiving anterior cortical input received peripheral input, and most neurons with posterior cortical input received peripheral input. Sixty per cent of reticular neurons showed the rhythmical firing closely related to the RJM. Therefore, these neurons may participate in masticatory pattern formation.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.