No abstract

Mice possessing two mutant alleles at the W or Sl locus are anemic and deficient in mast cells. These mouse mutants have black eyes and white hair. Because homozygous mutant rats at the newly found white spotting (Ws) locus were also black-eyed whites, the numbers of erythrocytes and mast cells were examined. Suckling Ws/Ws rats showed a severe macrocytic anemia and were deficient in mast cells. When bone marrow cells of normal (+/+) control or Ws/Ws rats were injected into C3H/He mice that had received cyclophosphamide injection and whole-body irradiation, remarkable erythropoiesis occurred in the spleen of +/+ marrow recipients but not in the spleen of Ws/Ws marrow recipients. When skin pieces of Ws/Ws embryos were grafted under the kidney capsule of nude athymic rats, mast cells did develop in the grafted skin tissues. Therefore, the anemia and mast cell deficiency of Ws/Ws rats were attributed to a defect of precursors of erythrocytes and mast cells. Because the magnitude of the anemia decreased and that of the mast cell deficiency increased in adult Ws/Ws rats, this mutant is potentially useful for investigations about differentiation and function of mast cells.

Introduction. Taka-amylase A (TAA) [EC 3.2.1.1 a-1,4-glucan 4-glucanohydrolase, Aspergillus oryzae] which was crystallized first by Akabori et al. in 19511) is a glycoprotein consisting of a single polypeptide chain of 478 amino acid residues with an amino (N)terminal alanine and a carboxy (C)-terminal serine.2~'3> The partial amino acid sequences of the N-and C-terminal regions,4~ '5> the carbohydrate chain structure° 7) and X-ray crystallographic analysis8> of the enzyme have been reported. This paper describes the complete amino acid sequence of TAA. Materials and methods. Crystalline TAA prepared from Takadiastase Sankyo9> was further purified by DEAE-cellulose column chromatography.10~ The homogeneous enzyme judged by polyacrylamide gel electrophoresis was reduced and carboxymethylated.'1 The cyanogen bromide cleavage at methionine residues of the reducedcarboxymethylated TAA (RCM-TAA) was performed in 70% formic acid for 24 h at room temperature. After removal of excess reagents by repeated lyophilization, the CNBr fragments were fractionated by gel filtration on a Sephadex G-75 column, by affinity chromatography on a Concanavalin A-Sepharose column, by ion-exchange chromatography on a SP-Sephadex C-25 column or an AG50W x 2 column and by paper electrophoresis. Methionine-containing peptides were isolated from tryptic and chymotryptic digests of maleylated RCM-TAA by a combination of gel filtration on Sephadex G-75 and G-50 columns, paper electrophoresis and high performance liquid chromatography. Amino acid compositions of peptides were determined with a Hitachi 8355 automated amino acid analyzer after hydrolysis of the samples with twice-distilled HCl containing 1 (v/v) phenol in evacuated sealed tubes at 110°C for 24, 48 and 72 h. Sequence analyses of the fragments were mostly performed by automated Edman degradation using a Beckman model 890C liquidphase sequencer in the presence of Polybrenel2) or an LKB 4020 College,

The two dissimilar composite polypeptide chains (A and B) in beta1-bungarotoxin were isolated as their reduced and carboxymethylated derivatives as reported in the preceding paper. The N-terminal sequences were determined with a sequenator up to the 39th residue for the RCM-A chain and up to the 25th residue for the RCM-B chain with repetitive yields of 90-95%. The tryptic and chymotryptic peptides from the two chains were isolated and their structures were determined by manual Edman degradation together with dansyl-Edman and carboxypeptidases A and Y. To complete the primary structures of the two chains, information on the tryptic peptides derived from the maleylated derivatives of the two chains was also used. The completed amino acid sequence of the A chain containing 120 residues (molecular weight, 13,500) is similar to that of notexin, a presynaptic neurotoxin from Australian tiger snake venom, and phospholipases A from other snake venoms. The amino acid sequence of the 60 residues in the B chain (molecular weight, 7,000) bears no resemblance to any basic polypeptides from snake venoms. The B chain probably plays a significant role by interacting with some components in presynaptic membranes of neurosmuscular junctions.

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.