Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated γ′ chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.

After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

     

Studies on organ specificity in experimental murine cryptococcosis

The chief histopathological features found in patients with cryptococcosis are both a cystic (gelatinous) lesion and a granulomatous reaction. These two tissue reactions are definitely different from each other, because a cyst is not accompanied with a significant cellular response, while a granuloma is formed as a result of various cell reactions. Therefore, it is very interesting that these two types of lesion can be observed in the same patient or in the same animal infected with Cryptococcus neoformans. From our previous paper (II) the authors reach such a thought that two steps may be required for the granuloma formation against C. neoformans infection: first, of phagocytosis by sessile macrophages of C. neoformans and second is related to T-cell function. This experiment was done to verify that the granulomatous response against C. neoformans infection might occur easily in the organs rich in sessile macrophages as compared with those poor in them and a polysaccharide capsule surrounding cryptococci may have effects to inhibit a migration of polymorphonuclear leucocytes or monocytes toward C. neoformans. C. neoformans strain RIB 12 (serological type A, mating type α) was used in this experiment. After a culture of a brain heart infusion glucose agar slant at 37 C for 3 days, yeast cells of the strain were harvested, and suspended in 1/15 M(p_H7.4) sterile phosphate buffered saline solution. Infective inoculum was prepared by adjusting the number of the yeast cells to 10⁵, 10⁶ or 5×10⁶/0.2 ml in a hemacytometer. Fourty-two male mice strain ddY were divided into 3 groups consisting of 14 each and one group was allotted to one of the cell suspensions. Each mouse was inoculated with 0.2 ml of the cell suspension into a tail vein and one mouse from each group was sacrificed at adequate intervals. At necropsies the brain, thymus, lungs, heart, liver, kidneys, spleen, pancreas, mesenteric lymph nodes, a part of the small intestine, testes and fat tissue were removed. From these organs histopathological sections, stained with HE or by PAS, were prepared. To investigate effects of a polysaccharide capsule to a migration of polymorphonuclear leucocytes or monocytes, double infections with C. neoformans and Aspergillus fumigatus, and an observation by the ‘Agar-Implantation method’ were done. As results, granulomata were formed easily in the organs rich in macrophages or lymphocytes such as the liver, spleen, lymph nodes, thymus, lungs, small intestine and fat tissue. On the contrary, in organs poor in the macrophages such as the brain, heart, pancreas, kidneys, adrenal glands and testes, the chief histopathological feature was a cyst formation containing numerous yeast cells. In the double infection, two types of lesions such as cysts and abscesses were observed in the sections of the brain. The former occurred against C. neoformans infection and the latter, against A. fumigatus infection. Even though a cyst was very close to an abscess, polymorphonuclear leucocytes or monocytes were never induced to C. neoformans. In the observation using the ‘Agar-Implantation method’, a severe cellular infiltration occurred to a perfect (teleomorphic) state of C. neoformans and very weak response, to yeast cells with a polysaccharide capsule. The difference may be due to the existence of the capsule, because a perfect state of C. neoformans is not surrounded by it.     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures I. Participation of structural changes of thylakoid membranes in light-induced 515-nm absorbance change

Light-induced absorbance change at 515 nm in spinach chloroplastswas studied in the temperature range from –2?C to 27?C.Lowering of temperature had no marked effect on the extentsof initial "light-on" spike and the steady-state change overthe temperature range examined, whereas the rate of recoveryof the 515-nm change was significantly reduced at lower temperatures.Above 15?C, recovery of the 515-nm change after continuous illuminationshowed a first-order kinetics. In contrast, the recovery wascomposed of a fast and a slow phases at lower temperatures. The fast phase of the recovery of the 515-nm change was acceleratedby carbonyl cyanide m-chlorophenylhydrazone, valinomycin plusK⁺ or sodium tetraphenylboron, while the slow phase was completelyeliminated in glutaraldehyde-fixed chloroplasts. Light-inducedchange in absorbance at 546 nm, an indicator of structural changesof membrane, showed almost the same dependency on temperatureas the slow phase of the recovery of the 515-nm change. Theseresults suggest that not only electric field formation acrossthe thylakoid membrane but also structural or conformationalchanges in the membrane participate in the 515-nm absorbancechange observed under steady illumination. (Received July 5, 1976; )     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures II. Relationship between 515-nm absorbance change and rapid H+ uptake in chloroplasts after a short flash illumination

The absorbance change at 515 nm induced by a short (7.6 µsec)light flash in spinach chloroplasts was studied at sub-roomtemperatures in relation to rapid H⁺ uptake into chloroplasts. Lowering of temperature caused a marked decrease in the rateof recovery of 515-nm absorbance change after a flash illumination.Initial rate of rapid H⁺ uptake, measured with absorbance changeof bromcresol purple (BCP), was also reduced at lower temperatures,in a parallel fashion. Half-recovery time of the absorbancechange at 515 nm and rise-time of the pH-indicating absorbanceincrease of BCP coincided well at each temperature studied.Values of the calculated activation energy for these two processeswere almost the same. The parallelism between the 515-nm absorbance change and therapid H⁺ uptake after a single flash illumination was also observedwhen the electric field decay and/or H⁺ translocation were acceleratedby ionophorous antibiotics, carbonylcyanide m-chlorophenylhydrazoneor phenazine methosulfate. From these results, it is suggestedthat the rapid H⁺ uptake into chloroplast is chemically coupledto electron transfer and at the same time diffusion- (or transport-)controlled. Membrane potential, reflected in the 515-nm absorbancechange is dissipated with the rapid H⁺ influx. A model for theelectron-transfer-coupled H⁺ translocation involving a plastosemiquinoneloop is presented. Dissipation of the illumination-formed inside-positivemembrane potential by the influx of H⁺ is explained by the model. (Received September 17, 1976; )     

Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of O2- with electron transfer components

Chemiluminescence of luminol (CLL) was induced by illuminatedspinach chloroplast fragments. CLL was diminished by superoxidedismutase or under anaerobic conditions and increased by anautoxidizable electron acceptor, methyl viologen. The optimumpH for CLL was 10.0-10.5. Ferredoxin and cytochrome c reducing substance (CRS) did notaffect the intensity of CLL, but accelerated the dark decayin the absence of methyl viologen. In the presence of methylviologen, ferredoxin and CRS lowered the intensity and acceleratedthe dark decay. 3-(4-Chlorophenyl)-1,1-dimethylurea diminishedCLL. Carbonylcyanide m-chlorophenylhydrazone accelerated theinitial rate of CLL increase at low concentration and inhibitedit at high concentration. Half-decay time of CLL after the cessationof light was shortened by inhibiting electron transfer on theoxidizing side of photosystem II. We conclude that most of the CLL observed in illuminated chloroplastsis dependent on O₂^–. The results also suggest that O₂^–is reduced by reduced ferredoxin or CRS and oxidized on theoxidizing side of photosystem II. The half life of O₂^–in illuminated chloroplasts was estimated from the half-decaytime of CLL to be a few sec. ¹ Present address: Kyushu Dental College, Department of Biology,Kitakyushu 803, Japan. (Received May 30, 1977; )     

Discrimination and characterization of photosystem I-and photosystem II-induced 515-nm absorbance changes in chloroplasts II. Separate local fields in thylakoids indicated by differential responses of the 515-nm absorbance change

Flash-induced 515-nm and 475-nm absorbance changes in spinachchloroplasts were investigated in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU). DCMU reduced the magnitude of the 515-nmabsorbance change by half and almost completely diminished theabsorbance change at 475-nm. The reduction of the 475-nm absorbancechange paralleled the inhibition of the photosystem II (PS II)light reaction. When chloroplasts were illuminated with red or far-red light,the ratio of A₅₁₅/A₄₇₅ changed depending on the photosystemactivated. Wide variations in the A₅₁₅/A₄₇₅ ratio observed insubchloroplast particle preparations were probably due to theenrichment and activation of one of the photosystems. We suggest that the photosynthetic pigments in the thylakoidmembrane are heterogeneously distributed, and chlorophyll bmolecules that may be responsible for the 475- nm absorbancechange are affected by the local field formed by the PS II lightreaction. On the other hand, an electric field due to the PSI reaction probably induced the absorbance change at 515-nm (Received February 24, 1978; )     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Synthesis of a polymide containing a glucose unit in the main chain

A new type polyamide containing a glucose unit in the main chain has been synthesized by the polymerization of C₁, C₃, C₄ blocked C₆-carboxymethylglucosamine, prepared from chitin. The deblocking procedure gave the water-soluble polyamide, of MW 1.5 × 10⁴, which can be regarded as a model for the recognition site of lectin.     

Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.