Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.     

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.     

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

Lateral distribution of phospholipid and cholesterol in apolipoprotein A-I recombinants

The influence of cholesterol on the assembly and structure of model high-density lipoproteins (HDL) has been investigated. Model HDL composed of apolipoprotein A-I (apoA-I) and 1,2-dimyristoylphosphatidylcholine (DMPC) formed spontaneously at the transition temperature (Tc) of the lipid. Those composed of apoA-I and 1-palmitoyl-2-oleoylphosphatidylcholine were formed by a cholate dialysis method. At low cholesterol/phospholipid ratios both lipids and assembly methods yielded a model HDL whose composition was identical with that of the initial mixture; as the cholesterol/phospholipid ratio of the initial mixture was increased, the fraction of cholesterol appearing in the model HDL decreased, and a negative correlation between the cholesterol and protein contents of the model HDL was observed. At high cholesterol/phospholipid ratios the association of apoA-I and phospholipids appeared to be thermodynamically unfavorable. The effects of cholesterol content on the thermal properties of a model HDL composed of DMPC and apoA-I were further investigated by differential scanning calorimetry, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, fluorescence energy transfer, and excimer fluorescence of pyrenyl derivatives of phosphatidylcholine (PC) and cholesterol. The addition of cholesterol decreased the transition enthalpy of DMPC, raised the midpoint of the transition, and modulated motional freedom in the phospholipid matrix. The amount of cholesterol required to produce these effects was lower in the model HDL than in multilamellar liposomes. In a model HDL composed of DMPC and apoA-I, the lateral diffusion of a pyrene-labeled cholesterol was dramatically changed at the Tc whereas little change was observed in that of a pyrene-labeled PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of baboon plasma high-density lipoproteins and of their major apoproteins.

Baboon high-density lipoproteins (HDL) were isolated by preparative ultracentrifugation between d = 1.063 and 1.215 g/mL. The HDL contains 48.8% protein and a lipid distribution similar to human HDL. The phospholipid distribution shows a low sphingomyelin value (5.9%), and the fatty acid composition of HDL is comparable to the human data except for the 18:1/18:2 ratio as a result of a higher 18:1 content in the CE and a lower 18:2 concentration in the PL. The major HDL apoproteins isolated on diethylaminoethyl-cellulose had a mobility on sodium dodecyl sulfate--polyacrylamide gel electrophoresis and a molecular weight and an amino acid composition similar to human apoA-I. However, the amino acid sequence of the first 30 residues of baboon apoA-I differed from the human apoprotein in residues 15 and 21. Treatment of apoA-I with carboxypeptidase A indicated a carboxyl-terminal sequence of Leu-Ser-Thr-Gln. Baboon apoHDL contained monomeric apoA-II with the mobility of monomeric human apoA-II and a molecular weight of 8500. The amino acid composition differed from the human apoA-II by the presence of arginine and by the absence of half-cystine and isoleucine. The circular dichroic spectra of apoA-I and apoA-II demonstrated a higher helicity compared to the human apoproteins. Recombination studies by microcalorimetry of apoHDL with dimyristoylphosphatidylcholine (DMPC) indicated similarities in the thermodynamic binding properties of the HDL apoproteins from man and baboon. The maximal-binding enthalpies of DMPC to apoHDL, apoA-I, and apoA-II were lower for the baboon than for the human apoprotein.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.