Amino acid sequence of beta-galactosidase. IX. Sequence of the central segment, CNBr peptides 10 to 17, residues 378 to 653.

The amino acid sequence in the 8 cyanogen bromide peptides comprising the central segment of beta-galactosidase is presented. This portion of the molecule, about 27% of the protein, contains over 40% of the lysine and tyrosine residues and has a slight excess of basic amino acids.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.     

Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components

Gel chromatography of kidney postmitochondrial fractions from control rats 2 hr after injection of ²⁰³Pb or after in vitro incubation with ²⁰³Pb disclosed the presence of two fractionated Pb-binding components plus binding in the void volume and total volume regions. The binding of Pb to the two components, with molecular weights of 11,500 and 63,000 daltons, was markedly decreased in Pb-pretreated rats. Sodium dodecyl sulfate-gel electrophoresis and autoradiography showed the presence of one major ²⁰³Pb band with an estimated molecular weight of 60,000 daltons. The 11,500-dalton peak did not incorporate ¹⁴C-leucine nor did concomitant administration of cycloheximide with the ²⁰³Pb inhibit incorporation of ²⁰³Pb activity, suggesting that the component is a preformed constituent of the kidney. In vitro incubation of brain, liver and lung postmitochondrial supernatants with ²⁰³Pb disclosed that these two binding components were also present in brain but not in liver or lung, suggesting a target tissue-specific localization for these Pb-binding macromolecules.     

Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Satellite III DNA: Regions of extreme endonuclease resistance and interindividual polymorphism in the Mb size range

Southern analysis of within-gel digested and restricted human cells has revealed very large Satellite III restriction fragments which show clear inter-individual length polymorphism. The Mb and sub-Mb length of these fragments indicate that they arise from regions of heterochromatin which contain homogeneous Satellite III sequences of peculiar resistance to common endonucleases. Based on sequence alone, such regions would be little digested by endonuclease digestion of chromatin in metaphase, regardless of its method of preparation. Polymorphic regions such as these might be expected to stain as part of the C-banding seen in endonuclease treated metaphase chromosomes, and may in part account for inter-individual C-band heteromorphisms.     

The interaction of the Escherichia coli mutD and mutT pathways in the prevention of A:T-->C:G transversions.

The Escherichia coli mutT mutator allele produces high frequencies of exclusively A:T-->C:G transversions. This is thought to be caused by a failure to prevent or remove A:G mispairs during DNA replication. The mutD5 mutator allele maps to the dnaQ locus which encodes the epsilon subunit of the DNA polymerase III holoenzyme. This subunit provides 3'-->5' exonuclease, proofreading, activity for removing mispaired nucleotides at the 3' end of the newly synthesized DNA strand. mutD5 has an altered epsilon resulting in reduced levels of proofreading and subsequent high mutation frequencies for all base-pair substitutions. We have analyzed the interaction between mutD5 and mutT-induced A:T-->C:G transversions by measuring reversion frequencies in mutD5 and mutT single mutator strains and mutD5mutT double mutator strains using the well-characterized trpA58 and trpA88 alleles. We find that the double mutator strains produce more A:T-->C:G substitutions than would be expected from simple additivity of the single mutator strains. We interpret this to mean that the two systems, at least in part, do act together to prevent the same mutational intermediate from producing A:T-->C:G transversions. It is estimated that over 90% of the mutT-induced A:G mispairs are corrected by proofreading at the trpA58 site while only about 30% are corrected at trpA88. Reversion frequencies in the mutD5mutT double mutator strains indicate A:G misincorporations occur about 100 x more frequently at trpA58 than at the trpA88 site. Using these and other data we also provide estimations of the fidelity contributions for mutT editing, proofreading and methyl-directed mismatch repair at the two trpA sites for both transversions and the transition that could be scored. In the case of A:T-->C:G transversions, both mutT editing and proofreading make major contributions in error reduction with mismatch repair playing a small or no role at all. For the A:T-->G:C transition, proofreading and mismatch repair were both important in preventing mutations while no contribution was observed for mutT editing.