Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization.

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.

Vitelline-coat lysin: comparison of lysins among three species in the genus Tegula.

Vitelline-coat lysins from two species of marine mollusc of the genus Tegula, Tegula lischkei and Tegula sp., were purified and their properties compared with those of Tegula pfeifferi. Cross-reaction tests among these three species proved that the lysin action on the vitelline coat was species specific. Each of the lysins from these three species is a single, basic polypeptide with a molecular weight of 16000-17000 and an isoelectric point of pH 10.5. All of them share a common antibody recognition site(s) for the anti-T. pfeifferi lysin antibody. Their amino acid sequences were analyzed using intact lysins and peptides separated by reverse phase high-performance liquid chromatography after V8 proteinase digestion. The amino acid sequences of the N-terminus (Nos 1-66) from the three species showed 98% homology, and those of the C-terminus (Nos 91-118) showed 89% homology. It was concluded that the species specificity of the vitelline coat lysin appears to be determined by a sequence of 24 residues (Nos 67-90) in the middle region of the molecule.

Ovulation stimulating substances in Drosophila biarmipes males: their origin, genetic variation in the response of females, and molecular characterization.

Ovulation responses of Drosophila biarmipes females to an injection of methanolic extract from conspecific males vary with the strains of females. This strain difference seems to be controlled by a small number of autosomal genes, with low responsiveness being recessive. Strangely, all D. biarmipes strains show a high level of ovulation after mating. We pursued the reason for this discrepancy and found that D. biarmipes males produce two different substances with ovulation-inducing activity. One of them is derived from the accessory glands and effective in females of all strains. Another originates in the ejaculatory duct and is inactive in some strains. In an active HPLC fraction of the ejaculatory duct extract, we found a peptide consisting of 32 amino acids. Its C-terminal region has a striking similarity to the sex-peptide of D. melanogaster, but the N-terminal region was entirely different. Evolutionary implications of these findings are discussed.

Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili.

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Isolation and structural determination of seminal vesicle-specific peptides of the terrestrial isopod, Armadillidium vulgare.

During the course of purifying the androgenic gland hormone of the terrestrial isopod, Armadillidium vulgare, that induces post-embryonic sex differentiation, four structurally related peptides were obtained and their structures determined by a combination of microsequence and mass spectral analyses. These peptides were found to exist speciffically in the seminal vesicle and vas deferens by a Western blot analysis, therefore being designated as seminal vesicle-specific peptides (SVSPs). They had essentially the same amino acid sequences but differed from one another in the truncation of several residues at the N-terminus and of one residue at the C-terminus, and in the modification of glutamine to pyroglutamate at the N-terminus. The longest peptides, SVSP-4, consisted of 60 amino acid residues with two intramolecular disulfide bridges. There is no significant homology with any other vertebrate or invertebrate peptides.

Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus.

Two proteins of molecular weights 20,000 (20K) and 15,500 (15.5K) are the major soluble substances released from the acrosomal vesicle of the abalone, Haliotis discus, spermatozoon. A crude preparation of them has been shown to possess lytic activity on the oocyte vitelline coat (VC). To elucidate the role(s) of each acrosomal protein (AP) in VC lysis, oocytes were examined after treatment with various AP preparations. The VC, which is about 1 micron thick, is composed of thin outer and inner electron-dense layers and a thick main layer of a fine filamentous feltwork. When oocytes were treated with a crude preparation containing both APs, the outer layer disappeared and the feltwork of the main layer loosened extensively. A preparation containing predominantly the 20K AP dissolved the outer layer completely and the main layer to some extent, whereas another preparation containing predominantly the 15.5K AP caused loosening of the main layer without alteration of the outer layer, suggesting that the 20K AP acts on the outer layer, whereas the 15.5K AP acts on the main layer. However, when purified, each AP by itself failed to dissolve the VC, although lysis occurred in a 1:1 mixture of these preparations. Moreover, when the oocytes were pretreated with the 20K AP and thoroughly washed, the 15.5K AP alone could induce lysis. These results suggest that the lysis of the outer layer requires both APs but not simultaneously. The 15.5K AP, which is located posteriorly in the acrosomal vesicle, must be released to act on the VC following the action of the 20K AP.

Structures and expression of mRNAs coding for major plasma proteins of Bombyx mori.

A group of structurally related proteins, referred to as '30K proteins', accumulate in a stage-dependent fashion in the larval hemolymph of the silkworm, Bombyx mori. To investigate the regulatory mechanisms of the 30K protein biosynthesis in the fat body, mRNA sequences coding for these plasma proteins were cloned and their structures and expression during the larval development were studied. Five plasmid clones, each bearing a distinct mRNA sequence coding for the 30K protein component, were isolated from a cDNA library constructed from the fat body poly(A)RNA of the mid-fifth instar larvae. These clones were remarkably similar to each other with respect to both the nucleotide and the amino-acid sequences. Each clone carried an open reading frame for amino-acid residues 251 to 264, with a typical signal sequence for transmembrane secretion at the deduced amino-terminal domain. Northern blot hybridization provided evidence that the developmental change in the amount of 30K protein mRNAs in the fat body reflects well that of the hemolymph concentration of 30K proteins, indicating that the biosynthesis of 30K proteins is developmentally regulated at a transcriptional or post-transcriptional level in the fat body.

Isolation and properties of androgenic gland hormone from the terrestrial isopod, Armadillidium vulgare.

The androgenic gland hormone (AGH) was purified from the male reproductive organs, including the androgenic glands, of Armadillidium vulgare by pH adjustment, ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and three kinds of high-performance liquid chromatography (HPLC). AGH consisted of two molecular forms, AGH I and AGH II. They were judged as homogeneous by HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectrofocusing, though their N-terminal residues were not detected. They seem to be monomeric proteins consisting of 157 and 166 amino acid residues, having molecular weights of 17,000 +/- 800 and 18,300 +/- 1000, and having isoelectric points of about 4.5 and 4.3, respectively. The activities of AGH I and AGH II were almost the same and their respective specific activities were 560 and 430 times that of the crude extract.

The complete amino acid sequence of vitelline coat lysin.

The vitelline coat lysin of a top shell, Tegula pfeifferi, is a single polypeptide consisting of 118 amino acid residues and having a relative molecular mass of 13800. The complete amino acid sequence of the vitelline coat lysin was determined by the analyses of five peptides obtained by cyanogen bromide degradation and three fragments obtained by Staphylococcus aureus protease digestion of the protein. The sequence showed the presence of microheterogeneities in the vicinity of the C-terminal half of the molecule and the existence of two homologous domain structures.