Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Electron Transfer between Plastocyanin and P700 in Various Types of Photosystem I Complex

Three types of PS I Chl-protein complex, PS I 180, PS I 65,and PS I 30, have been prepared and the kinetic properties ofthe transfer of electrons from plastocyanin to P700 in the PSI complexes with different sized antennae were examined. ThePS I 180 complex, which consists of 180 Chi per P700, showedthe almost same rate constant and effects of cations for thetransfer of electrons from plastocyanin to P700 as those obtainedwith PS I-enriched membrane fragments. The rate constant increasedwith the addition of low concentrations of monovalent and divalentcations, but decreased with high concentrations of cations.However, the rate was severely reduced in the case of the PSI 65 and PS I 30 complexes, and quite different effects of cationswere observed. Given the presence of additional 25- to 28-kDapolypeptides in the PS I 180 complex as compared to the PS I65 and PS I 30 complexes, we discuss a possible function forthese polypeptides in the regulation of the reaction betweenplastocyanin and P700. ¹This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Cultureof Japan. (Received May 27, 1988; Accepted November 7, 1988)     

Determination of Intracellular Chloride Ion Concentration in a Halotolerant Cyanobacterium Aphanothece halophytica

Salt inhibition of RuBP carboxylase activity from Aphanothecehalophytica was caused by Cl^-, but not by K⁺ nor Na⁺. The intracellularCl^- concentration increased about 4-fold from 35 mM to 150 mM,when NaCl concentration in the culture medium was increasedfrom 0.5 M to 2.0 M. ¹Permanent address: Department of Biochemistry, Faculty of Science,Chulalongkorn University, Bangkok, Thailand (Received February 12, 1988; Accepted June 1, 1988)     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.     

A dried preparation of liposomes containing muramyl tripeptide phosphatidylethanolamine as a potent activator of human blood monocytes to the antitumor state

Summary Studies were performed on the activation of human blood monocytes to the antitumor state by a dried preparation of multilamellar vesicle (MLV) liposomes in which synthetic muramyl tripeptide phosphatidylethanolamine (MTP-PE) was inserted directly into the liposome membrane. Dried liposomes composed of synthetic phospholipids [phosphatidylcholine (PC) and phosphatidylserine (PS) in a molar ratio of 7:3] were prepared by lyophilization. Dried liposome-MTP-PE was found to be superior in several ways to free desmethyl muramyl dipeptide (norMDP) or conventional liposome-MTP-PE, prepared immediately before use. First, dried lipsome-MTP-PE was stable and strongly activated monocytes when stored for over 3 months in a freezer at –°C or even in suspension at 4°C. Second, human monocytes in suspension, as well as in the adherent form, were activated to the tumoricidal state by interaction for at least 4 h with the dried preparation of liposome-MTP-PE. Third, monocytes activated with the dried liposome-MTP-PE or conventionally prepared liposome-MTP-PE maintained their tumoricidal activity for a longer period (4 days) than those activated with free norMDP. These results indicate that the dried preparation of liposome-MTP-PE can be stored for a long time, has a reproducible effect that can be standardized and should be valuable for in situ activation of human monocytes to the tumoricidal state, which is associated with eradication of cancer metastases.     

Distribution of Glycinebetaine in Old and Young Leaf Blades of Salt-Stressed Barley Plants

In barley plants exposed to stepwise salt-stress (up to 200mM NaCl), sodium and chloride ions accumulated preferentiallyin old rather than in young leaf blades. Furthermore, the levelof glycinebetaine in young leaf blades was approximately threetimes that in old leaf blades. ³Present address: Department of Biochemistry, Faculty of Science,Kasesart University, Bangkok, 10900 Thailand     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).