A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Purification and Characterization of Rice Embryo BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase)

Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984)     

Role of luteinizing hormone-releasing factor (LH-RF) and LH on maintenance of early gestation in rats.

The role of luteinizing hormone (LH) and LH-releasing hormone (LH-RH) in the maintenance of early pregnancy in rats was studied. Serum levels of progesterone (P) and LH were measured daily in untreated pregnant rats from Day 4 through parturition. Serum levels of P and LH were determined on Days 11 and 15 of pregnancy in animals treated with antisera to LH (LH-A/S) and to LH-RH (LH-RH-A/S) on Days 8-10. Serum levels of P peaked on Days 7 and 16 in untreated animals, after which they declined sharply just before delivery. Serum LH fluctuated between 30-160 ng/ml during pregnancy but did not exhibit any distinctive peaks. Treatment with .2 ml LH-A/S on Days 8-10 reduced serum P to virtually undetectable levels on Day 11, and only a slight recovery was evident on Day 15. Lower doses of LH-A/S had no effect. Administration of 1.3 ml LH-RH-A/S had no effect on serum levels of P or LH, and did not impede fetal development. The results indicate that LH is essential to the luteotropic complex of early pregnancy in the rat, and also that LH-RH-A/S can maintain to some extent basal levels of P and LH during early pregnancy.     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

Persistence of Repeated Sequences That Evolve by Replication Slippage

The evolution of short repeated sequences by replication slippage under the assumption of selective neutrality is modeled using a linear birth and death process. The equilibrium distribution, the distribution of the life expectancy of a repeated sequence when the process starts from two repeats, the age distribution of repeats, the probability of obtaining two genes with i and j copies which diverged t generations ago and the conditional variance of copy number given the repeat number is more than one are computed. The distributions of life expectancy and age are shown to have long tails. Also the statistic which estimates the conditional variance is shown to have a large coefficient of variation. Using these theoretical results, we develop an approximate test of our model and analyze persistent repeated sequences found in the primate beta-globin gene region and Oenothera chloroplast DNA which are polymorphic within species. We found one sequence in Oenothera chloroplast DNA which does not fit to our neutral model.     

Proteins and Carbohydrates in Xylem Sap from Squash Root

The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml^–1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 48–64 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992)     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.