Surface areas of 1-palmitoyl phosphatidylcholines and their interactions with cholesterol.

The activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow linear decrease in velocity as the concentration of NaCl was increased. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. The order of efficiency of added glucosyl acceptors in beta-glucosidase-catalysed reactions was found to be fructose greater than sucrose greater than glycerol greater than methanol. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated and an intimate glucosyl cation-carboxylate ion-pair intermediate and an alpha-glucosyl enzyme intermediate [Umezurike, G. M. (1981) Biochem. J. 199, 203-209]. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of beta-glucosidase in the presence of added glucosyl acceptors or of NaCl.     

Modulation of the membrane potential of the Schwann cell of the squid giant nerve fiber

1. The involvement of second messengers and of other chemical mediators, in the modulation of the membrane potential of the Schwann cell of the giant nerve fiber of the Tropical squid Sepioteuthis sepioidea is described. 2. The involvement of the cyclic nucleotide adenosine 3', 5' monophosphate (cAMP) in mediating the actions of the nicotinic Ach receptors of the Schwann cells is suggested. 3. The presence of octopaminergic receptors in the Schwann cells, mediating their actions through the activation of adenylate cyclase, is also described. 3. Receptors for vasoactive intestinal peptide (VIP) are also present on the Schwann cells, and their actions are mediated via a second messenger system that does not involve the activation of adenylate cyclase. 5. The three independent receptor systems referred above are able to interact in a complex way, which involves both their direct actions on the Schwann cell membrane potential and modulatory effects between the systems.     

Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions

We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions. A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides. Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands. In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment. High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction. The synthetic Holliday junction was found to be a specific inhibitor of a S. cerevisiae enzyme that catalyzes the cleavage of Holliday junctions. Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1. Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex. The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays.     

Proteolysis of apoprotein B during the transfer of very low density lipoprotein from hens' blood to egg yolk

We report an example of the enzymic cleavage of an apoprotein B (apoB), the main apoprotein in the very low density lipoprotein (VLDL) of laying hens' blood, in a normal biological process, the formation of egg yolk. Plasma VLDL was labeled in vivo with 3H-amino acids, isolated by centrifuging, and injected into another laying hen. Yolk VLDL was isolated and its apoproteins were separated. ApoB was not detected in this lipoprotein. Most of the label originally in apoB was distributed among four smaller yolk apoproteins, apovitellenins III to VI, which are a large proportion of the apoproteins of VLDL in yolk. This distribution of 3H suggested that 80% of apoB was cleaved at three places. One yolk apoprotein, apovitellenin II, was not labeled, indicating that it did not originate from an apoprotein in plasma VLDL. The site for cleavage of apoB in the ovarian tissue has not been determined, but cleavage may occur during receptor-mediated endocytosis. The pattern of cleavage of apoB during transfer to yolk was not imitated by some known proteolytic enzymes.     

Mouse glandular kallikrein genes. Structure and partial sequence analysis of the kallikrein gene locus

Mouse glandular kallikreins are encoded by a family of closely linked genes which are located on chromosome 7 at a site corresponding to the genetically defined Tam-1, Prt-4, and Prt-5 loci. We have characterized 24 kallikrein genes by genomic cloning and restriction mapping of 310 kilobase pairs of BALB/c mouse DNA. Most of these genes are highly homologous, have the same exon/intron organization, and are linked in clusters of up to 11 genes. Partial sequence analysis of the kallikrein genes has facilitated identification of those members of the family for which protein sequence data exist and assignment of those which are pseudogenes or encode proteins of unknown function. We find that a maximum of 14 mouse kallikrein genes have the potential to encode functional proteins.     

Identification and characterization of two novel lymphocyte function-associated antigens, L24 and L25

We describe the function and cell distribution of two novel cell surface antigens, L24 and L25. These antigens are broadly distributed on human lymphocytes. Monoclonal antibodies specific for these molecules block lysis by Class I- and II-specific cytotoxic T lymphocytes, but do not affect any other T cell functions tested. Anti-L24 antibody immunoprecipitates a molecule composed of two disulfide-linked monomers of 140 kd each. Anti-L25 antibody immunoprecipitates three proteins of 150, 85, and 75 kd. The study of these and other function associated molecules may provide insight into mechanisms of cytotoxic T lymphocyte recognition and/or function.     

Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.