Detection of side-chain proton resonances of fully protonated biosolids in nano-litre volumes by magic angle spinning solid-state NMR

We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C–13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 µg using a MAS frequency of 70 kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Hα resonances, in a single experiment providing a complementary approach to the established proton-detected amide-based multidimensional solid-state NMR experiments for the study and resonance assignment of biosolid samples, in particular for aromatic side chain resonances.     

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

Studies on the lipid and apolipoprotein compositions of two species of apoA-I-containing lipoproteins in normolipidemic males and females

Two species of apoA-I-containing lipoproteins (A-ILp), lipoprotein containing apoA-I and apoA-II (LpA-I/A-II) and lipoprotein containing apoA-I but no apoA-II (LpA-I), have been isolated from 20 normolipidemic adults (10 males and 10 females) by immunoaffinity chromatography. We have characterized the lipid and apolipoprotein compositions in these lipoproteins, and found sex differences. In A-ILp, the levels of lipids, except triglyceride, and the level of apoE were significantly higher in females than in males. In LpA-I/A-II, sex differences were found only in the levels of apoA-I and apoE. In LpA-I, the levels of all lipids, except triglyceride, and the level of apoA-I were significantly higher in females than in males. Therefore, sex differences observed in A-ILp appear to be due primarily to the differences found in LpA-I. Of considerable significance is our finding that the ratio of cholesteryl ester to total cholesterol in LpA-I was significantly lower than that in LpA-I/A-II in both males and females. This might suggest that LpA-I could be a carrier of free cholesterol.     

Receptor current fluctuation analysis in the labellar sugar receptor of the fleshfly

Fluctuations in the receptor current of the labellar sugar receptor of the fleshfly were analyzed. The receptor current was recorded extracellularly as a drop in potential between the tip and the base of the taste sensillum. After treatment with tetrodotoxin, the taste cells completely lost their impulses but retained their receptor currents, thus facilitating analysis of the receptor current without disturbance by impulses. The current fluctuation increased markedly when the sensillum was stimulated with effective sugars: maltose, sucrose, and fructose. The fluctuation increased in parallel with development of the receptor current, which indicates that it occurs as soon as the sugar reaches the apex of the sensory process. Analysis of fluctuations by computation of autocorrelation functions (ACFs) or power spectra (PS) revealed that: (a) the variance (mean square) of fluctuation vs. sugar concentration curve reached a maximum, in contrast to the monotonic increase shown by the receptor current; (b) the ACF was approximated by an exponential term, and its time constant differed according to the sugars used and their concentrations. The time constants for fructose and maltose decreased with increases in sugar concentration. At the concentrations of sugars evoking the same magnitude of receptor current, the time constant for fructose was the largest and that for maltose was the smallest. It was strongly suggested that transduction ion channels are present at the tip region of the sensory process of the sugar receptor cell and are operated directly by sugars.     

Genetic divergence in lizardfishes of the genusSaurida from southern Japan

Genetic divergence in four taxa of three species ofSaurida, S. undosquamis south (S) and north (N) types,S. wanieso andS. elongata caught from the East China Sea, Sea of Hyuga and Tosa Bay, was studied based on allelic frequencies at 23 genetic loci surveyed electrophoretically. Fixed allele substitution was observed at eight loci between the S and N types ofS. undosquamis and their genetic distance was 0.5582, within a range of differentiation at the species level. The S type ofS. undosquamis was found to inhabit the Sea of Hyuga and off Cape Ashizuri along the Kuroshio Current, in addition to the East China Sea. The low level of genetic variation found for this type was discussed in relation to its restricted habitat at the edge of the continental shelf. These factors, along with some morphological characters, indicate that the two types ofS. undosquamis should be recognized as distinct species.     

Circular dichroism studies on helical structure preferences of amino acid residues of proteins caused by sodium dodecyl sulfate

The extent of helical structure of 19 intact proteins and of 15 proteins with no disulfide bridges in the absence and presence of 10 mM sodium dodecyl sulfate (SDS) was determined using the curve-fitting method of circular dichroic spectra. The change in helicity caused by the addition of SDS was examined as a function of each amino acid fraction. An increase in the helicity upon the addition of SDS occurred in most of the proteins with no disulfide bridges (C proteins) and containing more than 0.06 Lys fraction. In most of the intact proteins (B proteins), most of which contained disulfide bridges, helicity in SDS decreased with an increase in Lys fraction. The helicity of the C proteins in SDS also tended to increase with an increase in the Leu and Phe fractions, while it decreased with an increase in the Gly fraction. For the helicity of the B proteins in SDS, there was a tendency to increase with increased Asn fraction and decrease with increased His fraction. On the other hand, amino acids were divided into eight groups according to their side-chain properties and the conformational preference for each of the amino acid groups of C proteins was calculated using a simple assumption.     

Electrochemical Behaviour of Copper-Containing Nitrite Reductase from Alcaligenes Faecalis Strain-6

Indirect and direct electrochemical reactions of copper containing nitrite reductase (NIR) from Alcaligenes faecalis strain-6 are described. The reactivity of mediators, including blue protein from the same organism (the native redox partner of NIR, AfBP), in electrocatalytic reactions (EC') involving a mediator, NIR and nitrite was investigated. Several types of EC were observed and AfBP was found to be an effective mediator in spite of its high redox potential. Direct electrochemistry was observed at an Indium Tin Oxide electrode (ITO) and an edge plane oriented pyrolytic graphite electrode (PGE). Observation of the redox activity of NIR at an ITO in an optically transparent thin layer electrode cell (OTTLE) showed that it underwent reversible changes in absorbance that corresponded to the applied potential. The electrochemically adsorbed NIR at PGE showed fast electrochemical kinetics in cyclic voltammetry. It is suggested that the weak affinity of NIR to the PGE electrode may prevent complete denaturation of NIR in the adsorbed state.     

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.