Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Human placental anticoagulant protein: isolation and characterization

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Absence of luminal bile increases duodenal content of cholecystokinin in rats.

The effects of the removal of bile from the proximal intestine on pancreas, plasma cholecystokinin (CCK) concentration, and duodenal content of CCK were examined in rats. Bile was excluded from the duodenum and introduced into the distal ileum through a silastic cannula for 7 days. Pancreatic juice was maintained to be normally secreted into the duodenum. After 7-day bile diversion, plasma CCK concentration and duodenal CCK content were significantly increased in bile-diverted rats. Trypsin content in the proximal intestine in bile-diverted rats was one-half that in control. Pancreatic wet weight, protein content, and DNA content in the pancreas were slightly increased, and lipase content was slightly decreased, by bile diversion, but none of these changes was statistically significant. Amylase content significantly decreased and chymotrypsin content significantly increased in bile-diverted rats. Intragastric administration of camostate (trypsin inhibitor) significantly increased plasma CCK concentration in both bile-diverted and control rats, and the net increase was much greater in bile-diverted rats than in control rats. In conclusion, bile diversion increased duodenal CCK content and increased the CCK response to luminal stimulant.     

A monoclonal antibody that recognizes sialyl-Lea oligosaccharide, but is distinct from NS 19-9 as to epitope recognition

A murine monoclonal antibody, designated as MSW 113, was generated using a human colonic cancer cell line, SW 1116, as the immunogen. MSW 113 was shown to be directed mainly to mucin-type oligosaccharide with sialyl-Lea antigens. The reactivity of MSW 113 to sialyl-Lea was stronger than that of NS 19-9, which is believed to be raised against the same determinant group. MSW 113 binds to sialyl-Lea-ol, LS-tetrasaccharide a, and disialyllacto-N-tetraose with higher affinities, compared to NS 19-9. These two antibodies could clearly be distinguished in that MSW 113 bound to sialic acid but not to fucose, whereas NS 19-9 bound to fucose but not to sialic acid. Thus, MSW 113 is directed more toward sialic acid-containing terminal structures while NS 19-9 is directed toward fucose-containing internal structures. MSW 113 was found to be useful for detecting antigens in the bloodstream of patients, especially those with pancreas cancer. Even NS 19-9 negative patient sera were positive for MSW 113.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.GalNAc transferaseHuman intestinal cancerous tissueBovine submaxillary gland mucin O-Glycosidically linked sugar chain     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.