Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Identity of soluble thiamin-binding protein with thiamin-repressible acid phosphatase in Saccharomyces cerevisiae

Two secretory glycoproteins of Saccharomyces cerevisiae, a soluble thiamin-binding protein and a thiamin-repressible acid phosphatase, were shown to be repressed to a similar extent by excess thiamin in the growth medium. Thiamin-repressible acid phosphatase was co-purified throughout the purification of the soluble thiamin-binding protein. Purified and deglycosylated soluble thiamin-binding proteins exhibited both thiamin-binding and acid phosphatase activity on non-denaturing polyacrylamide gel electrophoresis. Heat treatment of the purified soluble thiamin-binding protein caused a decrease in both activities with a similar inactivation profile. Furthermore, two thiamin-repressible acid phosphatase-defective mutants isolated had no and decreased soluble thiamin-binding activity, respectively. From the results, it was concluded that the soluble thiamin-binding protein is identical to the thiamin-repressible acid phosphatase in S. cerevisiae.     

Analysis of the replication mode of double minutes using the PCC technique combined with BrdUrd labeling

A cultured line of neuroblastoma cells (NB) was found to contain double minute chromosomes (DMs). DMs have been reported to be acentric and, therefore, to be segregated randomly into daughter cells without separating their sister elements. When NB cells were fused with Chinese hamster metaphase cells, prematurely condensed chromosomes (PCCs) were induced. DMs seen together with G₂ PCCs appeared to be closely paired, dot-like structures resembling DMs observable in metaphase cells. In contrast, DMs in G₁ cells showed a tendency to become single as the stage progressed so that the majority of DMs in late G₁ cells were actually no longer double. DMs in S-phase cells, however, again appeared double. These results clearly indicate why DMs are invariably double and never assume a quadruple configuration in metaphase cells in spite of their non-disjunctional segregation at anaphase. Such a characteristic mode of DM replication was further confirmed by a 5-bromo-2-deoxyuridine (BrdUrd) labeling experiment: when NB cells were exposed to BrdUrd for two successive rounds of DNA replication prior to PCC induction, half of the resulting single G₁ minutes as well as G₁ PCCs stained dark and the other half stained light after staining for sister chromatid differentiation.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

High-order structure of metaphase chromosomes: evidence for a multiple coiling model

When chromosome preparations made by the conventional air-drying method were processed with the OsO₄/TCH technique and examined by scanning electron microscopy (SEM), spiral structures in chromatids, which have been frequently observed to be present by light microscopy, were found to be composed of 30 nm fibres. In some portions these fibres appeared to be arranged in coils to form thicker fibres. When chromosome preparations were processed for SEM without air drying, chromosomes appeared to consist of fairly homogeneous thick fibrous structures measuring about 200 nm in diameter. In relatively condensed chromosomes, these 200 nm fibres appeared to be arranged perpendicular to the long axis of the chromatid. These findings suggest that chromatid spiral structures represent a regularly loosened state of the compactly spiralized 200 nm fibres which in turn consist of spiralized 30 nm fibres.     

Some properties of a Saccharomyces cerevisiae mutant resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole

A mutant of Saccharomyces cerevisiae highly resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole (2-aminohydroxyethylthiazole), an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), has been isolated. Its resistance to 2-aminohydroxyethylthiazole was about 10(4) times that of the sensitive parent strain. The amount of thiamin synthesized in the cells of the resistant strain grown in minimal medium was less than half of that of the sensitive strain. The ability to synthesize thiamin from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and hydroxyethylthiazole in the resistant strain was low compared with that of the sensitive strain. These results were found to be due to a deficiency of hydroxyethylthiazole kinase in the resistant strain: in sonic extracts of cells the enzyme activity was only 0.67% of that of the sensitive strain. Although the cells of the sensitive strain could accumulate exogenous hydroxyethylthiazole in the form of hydroxyethylthiazole monophosphate, no significant uptake of hydroxyethylthiazole by the cells of the resistant strain was observed. The possibilities that 2-aminohydroxyethylthiazole monophosphate may be the actual inhibitor of the growth of Saccharomyces cerevisiae, and that hydroxyethylthiazole may not be involved in the pathway of de novo synthesis of thiamin via hydroxyethylthiazole monophosphate, are discussed.     

Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Daphnia curvirostris Eylmann in Japanese high mountain waters (Cladocera: Daphniidae)

A species of Daphnia, Daphnia curvirostris Eylmann, found in high mountain lakes and ponds in central Japan is described. Although there were some differences in the shape of the male rostrum and the chromosome number between European populations as described by Johnson (1952) and Trentini (1980), and Japanese ones collected from high mountain waters, Japanese specimens had many characteristics similar to the taxon D. curvirostris of Europe.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Primordial germ cells and lymphomyeloid system in the embryos of the Aleutian skate,Bathyraja aleutica

Organogenesis in embryos (1 and 5 mm-disk widths) of the Aleutian skate,Bathyraja aleutica was described histologicaily in complete serial paraffin sections. Special attention was paid to localization of primordial germ cells (PGC) and development of lymphoid tissues. The embryo of 1 mm-disk width was at the somite stage with no evidence of organogenesis. PGC, gathered in the genital ridge, were seen in the somatic mesodermal layer as well as in the mesenchyme between the endoderm and splanchnic mesoderm of the 1 mm-embryo. Most of the visceral organs were developing in the embryo of 5 mm-disk width. With respect to the development of immune system, two pairs of thymus anlagen, filled with numerous thymic lymphocytes, were recognized in the pharyngeal region. A small focus of numerous immature blood cells appeared to be an anlage of the spleen was found between the stomach and the liver.