The carbohydrases of Bacillus stearothermophilus NCIB 11412 and NCIB 10814

Summary Two strains (NCIB 11412 and NCIB 10814) of the thermophilic organism Bacillus stearothermophilus were found to produce complex carbohydrase systems. The enzyme activities in each system include -amylase as the major component, maltase, pullulanase, a minor amylase and cyclodextrinase. The latter three activities are produced in low yield in both strains. A crude enzyme preparation from each strain possessed maltogenic properties on hydrolysis of soluble starch. Following rigorous purification procedures, the purified major -amylase from either strain did not produce maltose as a major end-product of starch hydrolysis. However, a partially purified mixture of pullulanase, minor amylase and cyclodextrinase activities from NCIB 11412 and NCIB 10814 produced 56.4% and 62.0% maltose, respectively, from soluble starch.     

PATMAT: a searching and extraction program for sequence, pattern and block queries and databases

A program has been developed that provides molecular biologistswith multiple tools for searching databases, yet uses a verysimple interface. PATMATcan use protein or (translated) DNAsequences, patterns or blocks of aligned proteins as queriesof databases consisting of amino acid or nucleotide sequences,patterns or blocks. The ability to search databases of blocksby ‘on-the-fly’ conversion to scoring matrices providesa new tool for detection and evaluation of distant relationships.PATMAT uses a pull-down, menu-driven interface to carry outits multiple searching, extraction and viewing functions. Eachquery or database type is recognized, reported, and the appropriatesearch carried out, with matches and alignments reported inwindows as they occur. Any of the high scoring matches can beexported to a file, viewed and recalled as a query using onlya few keystrokes or mouse selections. Searches of multiple databasefiles are carried out by user selection within a window. PATMATruns under DOS; the searching engine also runs under UNIX.     

Complete moles have paternal chromosomes but maternal mitochondrial DNA

Summary Complete hydatidiform moles contain only paternal chromosomes. To learn more of their origin, we used restriction endonuclease site polymorphisms found in the parental mitochondrial DNAs to demonstrate that moles contain exclusively maternal mitochondrial DNA. Thus, moles must arise from the fusion of one or two sperm with a mature but anucleate ovum.     

Isolation of a plant glycoprotein involved with control of intercellular recognition

A recognition molecule was isolated from stigmas of S-allele genotype S₂S₂ of Brassica oleracea var. capitata L. After Sephadex chromatography, it eluted as a single symmetrical peak during diethylaminoethane-cellulose chromatography. A high degree of purity was affirmed by: sedimentation as a single peak during ultracentrifugation through 5 to 20% sucrose gradients; elution as a single peak from Sephadex G-100; visualization as a single band which stains with Coomassie blue and periodic acid Schiff reagent after electrophoresis on polyacrylamide gels. Other criteria supporting the conclusion that it is a glycoprotein are: (a) the highly purified preparation is anthrone-positive and has a Lowry protein to anthrone-positive carbohydrate ratio of 1.3; (b) the preparation contains arabinose, galactose, glucose, and mannose, although it is not precipitated by concanavalin A; (c) the immunological properties of the molecule are lost following protease treatment, and it has a molecular weight of 90,000 by Sephadex gel-filtration analysis and 54,500 by velocity sedimentation analysis.     

Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes

Pulse-labeled oocyte proteins were found to have a maximum average half-life of 73 h. In general, larger peptides underwent degradation at a faster rate than smaller peptides. In this respect, oocytes are similar to most other cells. Microinjected ¹²⁵I-labeled bovine serum albumin (BSA) was degraded over a 40 h period with a half-life of 20–30 h, regardless of the method of protein labeling, culture medium employed, size of oocyte microinjected, or hormonal history of the oocyte. The last two results, if applicable to oocyte proteins in general, imply that protein catabolism is constant throughout the later stages of oogenesis and that growth is primarily regulated by a stimulation of anabolism. Individual proteins microinjected into oocytes undergo rates of degradation consistent with turnover rates obtained in other systems. Sequestered ¹²⁵I-labeled BSA is only partially (40%) degraded, which indicates that, unlike microinjected ¹²⁵I-labeled BSA, it has access to a cytoplasmic compartment (yolk platelets?) within which it is relatively stable.     

The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.