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)     

Absence of generalized immunosuppression in C57BL/6J mice implanted with Lewis T241 fibrosarcoma or Lewis lung carcinoma

Summary The immune status of C57BL/6J mice implanted with Lewis T241 fibrosarcoma or Lewis lung (LL) carcinoma was investigated on days 14 and 28 after implantation. Splenic lymphocyte responses were assessed in mitogen (Con A, LPS) mixed lymphocyte culture (MLC), natural killer (NK), graft-vs-host (GVH), and interleukin production assays. Except for NK-cell cytotoxicity, all other immunologic parameters were either comparable to those in medium-implanted controls or augmented. NK cytotoxicity was reduced in both tumor-bearing groups on day 28. The provision of NK potentiation therapy (-interferon, polyinosinic: polycytidylic acid) to T241 mice under various treatment conditions did not have any significant effect on lung metastasis or survival.The results of this study do not support the thesis that T241-or LL-bearing C57BL/6J mice are generally immunosuppressed. Indeed, when immune functions were assessed on the basis of total splenic activity, each of the measured immunologic parameters was substantially greater in animals with tumors than without. Further it seems improbable, considering the magnitude of the NK-cell defect in T241 mice on days 14 and 28 after implantation and the absence of a therapeutic response to NK-cell stimulants, that NK-cell cytotoxicity is intrinsically associated with resistance to tumor progression in this model.     

Formation of regular packets of Staphylococcus aureus cells.

Staphylococcus aureus, which usually forms grape-like clusters, has the ability to form regularly arranged cell packets. These regular cell packets are formed when the activity of its separation enzyme(s) is lost either by treatment with detergents, such as sodium dodecyl sulfate or Trition X-100, or by mutation of the cells. These cell packets consisted of 8 to 64 spherical cells that have a three-dimensional arrangement. Some irregularity in the arragement of cells in packets, however, can be observed by scanning electron microscopy. It is concluded that S. aureus fundametally divides along three definitely oriented planes that are located at right angles to each other. After cell division, the cells usually become translocated due to the action of a separation enzyme(s) to form grape-like clusters.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

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; )     

Establishment and characterization of a cell line from the American opossum (Didelphys virginiana)

A permanent tissue-cultured cell line (designated OK) has been established from kidney tissue of an adult American opossum. The OK line has been characterized with respect to morphology, chromosome constitution, tissue-culture requirements, and attainable mitotic arrest. The cells are epithelial-like with a stable nondiploid chromosomal modal number of 23. Cells grown in Eagle's minimal essential medium with 10% fetal bovine serum have a mean doubling time of 18 hr. The cell line OK is potentially useful for the isolation and purification of the mammalian X chromosome because of the size differential between the smaller X's and the larger autosomes.     

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.