In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP,and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.     

Amino acid sequence around the active site cysteine residue of calcium-activated neutral protease (CANP)

We have examined hydrophobic properties of Tetrahymena CaM using the uncharged probe, n-phenyl-1-naphthylamine (NPN) fluorescence. The maximal fluorescence intensity of Tetrahymena calmodulin (CaM) is less than 1/12 of that of the bovine brain CaM. In the phosphodiesterase activation, the potency of Tetrahymena CaM, which was represented by reciprocals of the quantity of CaM required for half-maximal activation of enzyme was 22.7% respectively, of that of the bovine brain CaM. Here, Tetrahymena CaM had less hydrophobic groups exposed in the presence of Ca2+. Then Ca2+-CaM dependent enzymes require much amount of Tetrahymena CaM, comparing with the bovine brain CaM.     

Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.     

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.     

Feedback regulation of bile acid biosynthesis in the rat

The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.     

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K_i values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.     

Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Molecular regulation of amino acid biosynthesis in plants

Summary Our understanding of amino acid biosynthesis in plants has grown by leaps and bounds in the last decade. It appears that most of the amino acid biosynthesis takes place in the chloroplast. Recent demonstration of glutamine synthetase and DAHP synthase in the vascular tisuue has added a new dimension in the complexity of the nitrogen cycle in plants. Isolation of various genes and transformation of plants with the modified forms of the genes are providing tools for understanding the regulation of various pathways. Plant transformation approaches are also going to provide the food of the future with an improved amino acid composition.     

Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].

Cysteine synthase [O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8] (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, I. Murakoshi [1992] Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress.     

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.     

Purification and characterization of Bombyx cysteine proteinase specific inhibitors from the hemolymph of Bombyx mori.

Protein inhibitors capable of inhibiting BCP (Bombyx cysteine proteinase) were found in the larval-pupal hemolymph of Bombyx mori. Two forms of the inhibitors, named BCPI (BCP inhibitor) alpha and BCPI beta, were purified from the pupal hemolymph by heat treatment and column chromatographies on CM-cellulose, Toyopearl HW-50, Phenyl-Sepharose, and Mono Q. Purified BCPI beta gave a single protein band with a molecular mass of 10,500 daltons on SDS-PAGE. BCPI alpha is mostly composed of the same molecular mass protein as BCPI beta. Both forms were inhibitory towards other cysteine proteinases such as cathepsins L,B and papain but had no effects on trypsin and pepsin. Both forms inhibited the processing of the enzymatically inactive proform of BCP (pro-BCP) to the activated mature BCP. BCPI alpha and BCPI beta shared many other features such as molecular mass determined by gel filtration, antigenicity, and HPLC profiles. NH(2)-terminal amino acid sequencing of the purified inhibitors revealed that three amino acid residues were different in the BCPI alpha and BCPI beta sequences, all others being identical. The hemolymph BCP inhibitor increased activity approximately four- to fivefold at the time of spinning and maintained this level of activity during pupation.