Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.     

The effects of a known family-size distribution on the estimation of genetic parameters.

We consider the question: In a segregation analysis, can knowledge of the family-size distribution (FSD) in the population from which a sample is drawn improve the estimators of genetic parameters? In other words, should one incorporate the population FSD into a segregation analysis if one knows it? If so, then under what circumstances? And how much improvement may result? We examine the variance and bias of the maximum likelihood estimators both asymptotically and in finite samples. We consider Poisson and geometric FSDs, as well as a simple two-valued FSD in which all families in the population have either one or two children. We limit our study to a simple genetic model with truncate selection. We find that if the FSD is completely specified, then the asymptotic variance of the estimator may be reduced by as much as 5%-10%, especially when the FSD is heavily skewed toward small families. Results in small samples are less clear-cut. For some of the simple two-valued FSDs, the variance of the estimator in small samples of one- and two-child families may actually be increased slightly when the FSD is included in the analysis. If one knows only the statistical form of the FSD, but not its parameter, then the estimator is improved only minutely. Our study also underlines the fact that results derived from asymptotic maximum likelihood theory do not necessarily hold in small samples. We conclude that in most practical applications it is not worth incorporating the FSD into a segregation analysis. However, this practice may be justified under special circumstances where the FSD is completely specified, without error, and the population consists overwhelmingly of small families.     

Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy

Pulsed electron paramagnetic resonance spectroscopy has been used to detect Mn(II)-ligand superhyperfine couplings in complexes with creatine kinase and in the Mn(II) metalloprotein concanavalin A. Electron spin-echo envelopes from Mn(II), bound in these complexes, are modulated by superhyperfine interactions between Mn(II) and nearby, weakly coupled nuclear spins. The characteristic frequencies of the modulations were obtained by Fourier transformation of the three-pulse, spin-echo envelopes. In transition-state analogue complexes of creatine kinase (enzyme-MnIIADP-anion-creatine), superhyperfine interactions from the directly coordinated nitrogen of the thiocyanate ligand give envelope modulations. The source of the modulations was confirmed by measurements with the 14N and 15N forms of thiocyanate. On the other hand, the nitrogen of coordinated nitrate, which is two bonds removed from the paramagnetic center, does not produce detectable modulations. In spectra for Mn(II) concanavalin A, envelope modulations are detected due to the nitrogen of the coordinated histidine residue. Complexes prepared in 2H2O give strong signals due to weakly coupled 2H. For Mn(II)-doped single crystals of sodium pyrophosphate, signals are observed in the frequency domain spectra that are due to coupling from 31P. Phosphorus signals from the ADP ligand in complexes with creatine kinase show approximately the same coupling constant but have a much broader line width.     

Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein

SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

Comparison of male adult population densities of the oriental and artocarpus fruit flies,Dacus spp. (Diptera: Tephritidae), in two nearby villages in Penang,Malaysia

The populations of native male adult oriental fruit fly Dacus dorsalis (Hendel ) and artocarpus fruit fly D. umbrosus (F.) in two selected site (BU and SD) were estimated weekly by the capture-recapture technique using live traps baited with methyl eugenol. In BU where many varieties of fruit trees were grown, the estimated population densities of D. dorsalis were between 980 and 3100 male flies per ha between May and July, 1984. During the same period, in SD where there were fewer number and varieties of fruit trees, the estimated population densities were between 300 and 1000 flies per ha. The estimated population densities of D. umbrosus over the same period were between 570 and 1290 flies per ha in BU; and between 5 and 95 flies per ha in SD. Of a total 6828 marked D. dorsalis flies released only one fly (released 6 weeks earlier in BU) was caught in a different site.     

A histological study of the hypophysial-gonadal system during sexual maturation and spawning in the milkfish, Chanos chanos (Forskal)

The pituitary gland of the milkfish, Chanos chanos , was studied at different stages of sexual maturation and spawning. Consecutive median sagittal sections were treated with a range of stains to demonstrate the different cell types and regions. The milkfish pituitary consists of a neural component, the neurohypophysis, and an epithelial component, the adenohypophysis, which in turn consists of three regions: the rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). However, unlike most teleosts, the pituitary gland of the milkfish is encased in a bony chamber, has dorsal and ventral lobes and extends anteriorly from its point of origin at the base of the brain. PAS (+) basophils are found in all regions of the adenohypophysis, but mostly in the proximal pars distalis. These cells undergo hypertrophy and hyperplasia during sexual maturation, shrinkage and degranulation during spawning.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

新疆石河子地区印度麻花叶病植株病毒分离物的鉴定

1984年,从新疆石河子农学院实验站印度麻花叶病植株上,分离到一株病毒分离物Sc-1,经汁液摩擦接种试验表明,它可以侵染10种豆科植物和2种藜科植物。在印度麻、蚕豆、豌豆、箭舌豌豆、扁豆、山藜豆、田菁和红三叶草上引起系统花叶,在豇豆上产生局部枯斑和系统花叶,在苋色藜、昆诺藜上表现为系统黄斑。失毒温度为55～60℃,稀释限点10~(-3)～10~(-4),体外保毒期3～4天。可经汁液和蚜虫传播,不通过种子传毒。病毒粒体为线条状,大小为13～15×750nm。光学显微镜检查可见,病叶表皮细胞内形成不定形的内含体。电镜下可见风轮状、环状内含体。分离物Sc-1与菜豆黄色花叶病毒(BYMV)抗血清呈阳性反应。我们将Sc-1归为菜豆黄色花叶病毒(BYMV),且为豇豆株系。