Yukiho Kubota

Human cellular src gene product: Identification of the myristoylated pp60c-src and blockage of its myristoyl acylation with N-fatty acyl compounds resulted in the suppression of colony formation

A p60K protein in human colon adenocarcinoma tumor cell lines was identified as a myristoylated pp60c-src by fluorography and radioimmunoprecipitation analysis. Prevention of the myristoylation of pp60c-src was determined with N-fatty acyl glycinal compounds. Of the compounds tested, N-myristoyl glycinal diethylacetal, N-lauroyl glycinal diethylacetal, N-myristoyl glycyl glycinal diethylacetal, and N-myristoyl-4-aminobutyl-aldehyde diethylacetal strongly blocked the myristoylation, but N-decanoyl glycinal diethylacetal and N-palmitoyl glycinal diethylacetal did not. The myristoyl blocking compounds depressed colony formation, cell proliferation, and specific localization to the plasma membrane of pp60c-src. The results taken together suggest that myristoylation of the c-src oncogene product may be very important for tumorigenicity of c-src gene expressed cells.

Antimyristoylation of the gag proteins in the human immunodeficiency virus-infected cells with N-myristoyl glycinal diethylacetal resulted in inhibition of virus production.

The gag proteins of human retroviruses such as human immunodeficiency virus (HIV-1) are specifically myristoylated in their amino termini (1, 2, 3). N-myristoyl glycinal diethylacetal (N-Myr-GOA) and other N-Myr-compounds (N-Myr-Gly-GOA, N-Myr-Gly-Gly-GOA and N-Myr-Gly-Gly-Gly-GOA) were newly synthesized and investigated for activity of antimyristoylation of these gag proteins and for influence on viral replication. Of the N-Myr-compounds tested, N-Myr-GOA most severely inhibited the protein myristoylation; N-Myr-Gly-GOA also inhibited it, but moderately. Furthermore, it was observed that N-Myr-GOA at 20 microM caused noticeable inhibition (about 80%) of the production of mature HIV in the HIV-1-infected MT-4 cells. In this system, N-Myr-GOA substantially inhibited more than 90% of the N-myristoylation of p17 gag protein produced in the HIV-1-infected MT-4 cells. These results suggest that the N-myristoylation of p17 gag protein of HIV-1 may be essential in its structural assembly or maturation.

Electrophoretic and spectroscopic analyses of equine α2-macroglobulin with cleavage of the thiol ester bonds by methylamine

Reaction of equine alpha 2-macroglobulin (alpha 2M) with methylamine caused generation of 3.7 mol of thiol groups per mole of the protein, and the second-order rate constant of the generation was calculated to be 3.5 M-1 s-1. The inhibitory profile of caseinolytic activity of trypsin indicated that one molecule of equine alpha 2M inhibited two molecules of trypsin, similar to human alpha 2M. The methylamine-treated equine alpha 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human alpha 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine alpha 2M is substantially unperturbed by cleavage of the thiol ester bonds and that the intact thiol ester bonds per se are not essential for the ability of equine alpha 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine alpha 2M showed that this treatment caused only a small change in conformation of the protein. Reaction of the methylamine-treated protein with trypsin induced appreciable changes in the spectra, indicating a large change in conformation of the protein. These findings were consistent with the results obtained by electrophoresis: The band of methylamine-treated equine alpha 2M showed indistinguishable mobility from that of the unmodified protein, indicating that no appreciable change in conformation occurred, and distinctly different mobility from that of the unmodified or methylamine-treated equine alpha 2M when each had reacted with trypsin.

The functional site of placental anticoagulant protein: Essential histidine residue of placental anticoagulant protein

Placental anticoagulant protein (PAP) rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH 7.9 and 8 degrees C. Photooxidized PAP failed to bind the phospholipid vesicle. It seemed unlikely that the protein underwent a change in molecular size during the photooxidation on the basis of its behavior in electrophoresis and gel filtration. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. The peptide, SHLRKV, was included in the functional site of PAP and still showed an anticoagulant activity. On the other hand, the peptide which substituted histidine by alanine, SALRKV, no longer showed the activity. It was shown that the histidine residue is involved in Ca2+ or the phospholipid binding site of the protein.

Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells.

Novel antibodies were raised against a synthetic NH2-terminal myristoyl glycine moiety which is characteristic of N-myristoyl-proteins. Antisera raised against N-myristoyl-Gly-hemocyanin reacted with N-myristoyl-Gly-[125I]albumin. The immunoreaction was competed for by albumin conjugated with N-myristoyl-glycine, while underivatized albumin had no effect. Of the [3H]myristate-labeled proteins detected, pp60v-src, which is a transforming protein of Rous sarcoma virus, and p19gag and p17gag, which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N-myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.

A novel antibody against a synthetic NH2-terminal myristoyl glycyl src peptide can detect pp60v-src, the transforming protein of Rous sarcoma virus

Novel antibodies were raised against a synthetic NH2-terminal myristoyl(Myr-) tetrapeptide(N-Myr-Gly-Ser-Ser-Lys) which is characteristic of an NH2-terminal portion of pp60v-src, the transforming protein of Rous sarcoma virus. Antisera raised against N-Myr-Gly-Ser-Ser-Lys-haemocyanin reacted with 125I-albumin conjugates with N-Myr-Gly-Ser-Ser-Lys. The immunoreaction was competed for by haemocyanin as well as albumin conjugated with this N-Myr-peptide, while underivatized proteins or an NH2-terminal octapeptide (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys) had no effect. N-Myr-Gly-Ser-Ser-Lys-(125I)tyramine was also recognized by the antibody. The reaction was competed for by N-Myr-Gly-Ser-Ser-Lys, but not by Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys. These results suggest a high affinity of the antibody for an N-Myr-peptide moiety. The major (3H)myristate-labelled protein of an apparent molecular weight of 60,000 was detected from chick embryo fibroblasts transformed by Rous sarcoma virus (tsNY68). This protein was demonstrated to possess N-Myr-Gly-Ser-Ser-Lys by the immunoprecipitation and HPLC analyses. Furthermore, the entire circumference of the transformed cells was stained by the antibody upon an immunofluorescent microscopic observation. Thus, these results taken together indicate that the haptenic antibody raised against the myristoyl peptide is useful to detect pp60v-src protein.

Myristoylated src-oncogene products are potently detected by the monoclonal antibody to the NH2-terminal myristoyl-Gly-Ser-Ser-Lys src-peptide.

Monoclonal antibodies were raised against a synthetic NH2-terminal myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Ser-Lys) which is characteristic of an NH2-terminal portion of pp60src, the transforming protein of src-oncogene. The antibody reacted with the albumin conjugated with both the N-myristoyl and N-lauroyl-tetrapeptides, but concentrations at which 50% of the immunoreaction was inhibited were 5 pmol for the N-myristoyl and 830 pmol for N-lauroyl tetrapeptidyl albumin. On the other hand, N-palmitoyl tetrapeptidyl and underivatized albumin, and Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys octapeptide had no effects. These results suggest a high affinity of the antibody for an N-myristoyl-Gly-Ser-Ser-Lys moiety. src-Oncogene products in Rous sarcoma virus-transformed cells and human colon carcinoma tumor cells were selectively identified as myristoylated pp60src by immunoprecipitation analyses with the antibody.

Effect of elastase on glucose and lipid metabolism in rat fat cells.

We examined the effects of elastase [EC 3.4.21.11] on lipogenesis, antilipolysis, and pyruvate dehydrogenase activity in rat epididymal adipose tissue in comparison with those of insulin and trypsin [EC 3.4.21.4]. The rate of conversion of [3-3H]-glucose into lipid in fat cells was stimulated by elastase, trypsin, and insulin. When fat pads were incubated with elastase, trypsin, or insulin in the presence of glucose, pyruvate dehydrogenase activity in the homogenate of the incubated fat pads was markedly increased. In the absence of glucose, elastase did not increase pyruvate dehydrogenase activity, though trypsin and insulin showed a slight but significant increase. Further, the increasing effect of elastase in the presence of glucose was inhibited by the addition of 3-O-methylglucose or phlorizin to the incubation mixture of the fat pads. Trypsin and insulin still showed a significant increase under similar conditions. When the homogenate of intact fat pads was incubated with elastase, the pyruvate dehydrogenase activity was progressively decreased with increase in the concentration of elastase. Concanavalin A showed an additive effect on the pyruvate dehydrogenase activity increase caused by elastase, whereas such an effect was not observed with insulin or H2O2. The stimulation of lipolysis by epinephrine in the fat cells was not suppressed by elastase, in contrast to trypsin and insulin. These results suggest that elastase reacts with the cell surface, facilitates glucose transport into the fat cells, and consequently affects glucose and lipid metabolism by somewhat different mechanisms from those of insulin and trypsin.