Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aid for the Choice of Buffer Concentration

The peptic hydrolysis of bovine β-lactoglobulin (β-Lg) was performed to establish the basis for producing a low-phenylalanine peptide rather than a free amino acid mixture for use in the dietetics of phenylketonuria. A 1% β-Lg solution (pH 1.5) was incubated with 0.01% pepsin at 37°C for 24 hr. The peptides produced were fractionated by high-performance liquid chromatograhy to analyze their constituent amino acids. Most of the major peptides were identified in the light of the primary structure of α-Lg to assign 31 cutting points in their protein molecule. These included cutting points at the carboxyl side of Phe-82, Phe-105 and Phe-136. This result suggests that further hydrolysis of the peptic hydrolysate of β-Lg with an exopeptidase, particularly with a carboxypeptidase, would be effective in liberating phenylalanine to produce a low-phenylalanine peptide mixture.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Electrophoretic characterization of ribosomal subunits and proteins in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells.

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.     

Natural development time of Eodiaptomus japonicus (Copepoda: Calanoida) in Lake Biwa

Seasonal and ontogenetic changes in natural development timewere studied for Eodiaptomus japonicus in Lake Biwa in 1986and 1987. Wild individuals in a certain developmental stagewere collected, fed on natural food and examined until theyhad moulted twice. Natural development times fluctuated irrespectiveof temperature from May to October. Food deficiency delayeddevelopment in all feeding stages, and food availability probablydetermined natural development time. Serious food limitationraised mortality in copepodid stage I. In November developmentwas delayed even with enough food. The development of E.japonicuswas almost isochronal except for a short prefeeding naupliarstage I and a long copepodid stage V.     

Analysis of the microheterogeneity of the glycan chain of rat transferrin

To investigate the microheterogeneity of the glycan chain of rat transferrin, either the protein moiety was labeled with 125I or the sialyl residues with 3H. The molecule was then subjected to Con A chromatography. Three components were obtained. Each was enzymatically desialylated and sialyl/protein molar ratios were calculated. The native protein as well as the 3 components were also subjected to isoelectric focusing. The results indicated that rat transferrin may have 3 types of glycan chain: The major type (60%) corresponds to a molecular species with triantennary branching, while 30% consists of molecules with biantennary and 10% with tetraantennary branching. The last species has not been previously described.     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)