Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of¹⁴C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.     

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.     

Enzymatic O-glycosylation of kappa-caseinomacropeptide by ovine mammary Golgi membranes

The results of subcellular fractionation of sheep mammary gland membranes indicate that N-acetylgalactosaminyl polypeptide transferase and galactosyl-N-acetylgalactosaminyl transferase, which are involved in the assembly of disaccharide units of kappa-casein, are localized chiefly in Golgi membranes. The glycosyltransferase activities incorporating N-acetyl [1-14C] galactosamine and [U-14C] galactose from uridine diphosphate N-acetyl [1-14C] galactosamine and uridine diphosphate [U-14C] galactose, respectively, were measured after membrane solubilization with Triton X-100 either with unglycosylated caseinomacropeptide, or with this polypeptide containing the N-acetylgalactosamine side chain residues (desialylated and degalactosylated caseinomacropeptide). Radioactive N-acetylgalactosamine was incorporated in the unglycosylated acceptor peptide, and the glycosidic bonds in the product were alkali labile, suggesting that they were linked to the hydroxyamino acid residues. In addition radioactive N-acetylgalactosamine was released after alpha N-acetyl-D-galactosaminidase treatment of labelled caseinomacropeptide. [U-14C] galactose was incorporated in the desialylated and degalactosylated acceptor peptide. Reductive alkaline treatment of [U-14C] galactose peptide resulted in the release of a major product, the chromatographic properties of which in TLC were identical with authentic galactosyl (1 leads to 3) N-acetylgalactosaminitol. The structure of the labelled disacchariditol determined after periodate oxidation (two equivalents) by gas liquid chromatography-mass spectrometry revealed that the [U-14C] galactose was linked to position C-3 on the N-acetylgalactosaminyl-residue. The anomery of the galactose, as determined by a chemical method, indicates unambiguously a beta configuration.