Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by B?chinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.     

Localization of lipid peroxidation products in liposomes after γ-irradiation

The distribution of lipid peroxidation products in liposomes after γ-irradiation at various doses was studied. Increases in thiobarbituric-acid-reactive substances, in the absorbance at 232 nm and in hydroperoxides were observed mainly in liposomal membranes after relatively low doses of irradiation, while carbonyl compounds were distributed both inside and outside the membranes. After higher doses of irradiation, however, the absorbance at 232 nm and the amount of hydroperoxides reached a maximal level in the membrane portion and then decreased when the decomposition products were released from the membranes. Under this condition, malondialdehyde and other carbonyl compounds were increased mainly in the medium of liposomal suspension. These results are discussed with reference to the lipid peroxidation process which is induced quantitatively by ionizing radiation.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol.

Polyethylene glycol (PEG) stimulates ligation with T4 DNA ligase. In 10% (w/v) PEG 6,000 solutions, only intermolecular ligation is enhanced by monovalent cations, while both inter- and intramolecular ligation occur without their presence. Similar stimulation was also caused by divalent cations or polyamines in the PEG 6,000 solutions. Such properties of the ligase could be applied to control the extent of inter- and intramolecular ligation. Ligation with cations or polyamines in 10% PEG 6,000 solutions was effective for intermolecular ligation. Ligation without cations or polyamines in 6.0% to 10% PEG 6,000 solutions was effective for intramolecular ligation.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.     

Reconstitution studies on the involvement of radiation-induced lipid peroxidation in damage to membrane enzymes

The effect of radiation on the drug-metabolizing enzyme system of microsomes, reconstituted with liposomes of microsomal phospholipids, NADPH-cytochrome P-450 reductase and cytochrome P-450, was examined to elucidate the role of lipid peroxidation of membranes in radiation-induced damage to membrane-bound enzymes. The reconstituted system of non-irradiated enzymes with irradiated liposomes showed a low activity of hexobarbital hydroxylation, whereas irradiated enzymes combined with non-irradiated liposomes exhibited an activity equal to that of unirradiated controls. Irradiation of liposomes caused a decrease in cytochrome P-450 content by destruction of the haem of cytochrome P-450 and also inhibited the binding capacity of cytochrome P-450 for hexobarbital. The relationship between radiation-induced lipid peroxidation and membrane-bound enzymes is discussed.     

Apparent dipeptidyl peptidase activities of acylamino acid-releasing enzymes

An acylamino acid-releasing enzyme purified from porcine liver showed peptidase activity above pH 8. Of the non-acylated peptides tested, this peptidase activity was only exerted on peptides with Gly or Ala at their N-termini. These results are consistent with the previous observations for similar enzymes from sheep red blood cells (Witheiler, J. & Wilson, D.B. (1972) J. Biol. Chem. 247, 2217-2221) and beef liver (Gade, W. & Brown, J.L. (1978) J. Biol. Chem. 253, 5012-5018). The pH dependence of the peptidase activity showed that only peptides with uncharged N-terminal amino acids such as glycyl- or alanyl-peptides act as substrates for the enzyme. These results suggest that the peptidase activity seen for the acylamino acid-releasing enzyme is an intrinsic activity of the enzyme that is triggered by misrecognition of uncharged smaller N-terminal amino acids in non-acylated peptides as acyl groups at higher pHs.