Interactions of troponin subunits: free energy of binary and ternary complexes

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT.TnI [TnC.(TnT.TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT.TnI. The free energies were -9.0 kcal.mol-1 for TnC.TnT, -9.2 kcal.mol-1 for TnT.TnI, and -8.7 kcal.mol-1 for TnC.(TnT.TnI). In the presence of Mg2+ the free energies of TnC.TnT and TnC.(TnT.TnI) were -10.3 and -10.9 kcal.mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal.mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT.TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal.mol-1, -18.9 kcal.mol-1, and -21.6 kcal.mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for active half-molecules of alpha 2-macroglobulin formed by dissociation in urea

Urea caused dissociation of alpha 2-macroglobulin (alpha 2M) into half-molecules (two disulfide-bonded subunits) as revealed by gel electrophoresis. The fraction of whole molecules remaining decreased with increasing urea concentration. Half-dissociation occurred at about 2.2 M. The ability of alpha 2M to inhibit trypsin also decreased with increasing urea concentration, but the activity-urea curve was shifted to the right as compared to the dissociation-urea curve. Thus, at 3 M urea, gel electrophoresis showed only 6.6% whole molecules, whereas the trypsin inhibitory activity was 95% of that in buffer with no urea, suggesting that half-molecules retain activity. In addition, complexes formed in urea with 125I-labeled trypsin were observed to migrate as half-molecules even though only 50% of such complexes were covalent. These results are surprising in light of the report by Gonias and Pizzo [Gonias, S., & Pizzo, S. (1983) Biochemistry 22, 536-546] that half-molecules formed by mild reduction are active; reduction is assumed to divide the molecule along an axis orthogonal to the break caused by urea. This suggests that active half-molecules can be formed by splitting either the covalent or noncovalent bonds that hold the subunits together. A model is proposed that can account for this possibility. It has the same dimensions and symmetry as a previous model of Feldman et al. [Feldman, S.R., Gonias, S.L., & Pizzo, S.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704] and accounts in a similar way for previous functional studies of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS-4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15- to 16-fold for FS-4 cells, as opposed to 3- to 4-fold typically obtained in a batch culture.     

Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

Adsorption of ionized and neutral pentachlorophenol to phosphatidylcholine membranes

We have studied adsorption of pentachlorophenol (PCP) to phosphatidylcholine (PC) membranes by measuring the electrophoretic mobility of multilayered lipid vesicles in PCP solutions. PC vesicles become negatively charged due to the adsorption of ionized PCP, and we have found that their zeta potential depends upon the ionic strength and pH of the aqueous suspension. We have shown that the experimental results can be adequately accounted for in terms of a two-component Langmuir-Stern-Grahame adsorption model assuming that the 'PCP adsorption sites' are occupied either by the neutral (HA) or the ionized (A-) species. The characteristics of adsorption isotherms of the PCP - PC membrane are as follows: the association constants are KA = 55,000 dm3/mol, KHA = 279,000 dm3/mol; 4.3 PC molecules make up each PCP adsorption site at saturation; the linear partition coefficients are beta HA = (15.5 +/- 0.7) x 10(-5) m and beta A = (3.0 +/- 0.3) x 10(-5) m. The properties of PCP adsorption isotherms for PC membranes predict an increased pKa value of membrane-bound PCP, which has been observed in related studies.     

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

Optimization by simulating molecular evolution

Based on the analogy between mathematical optimization and molecular evolution and on Eigen's quasi-species model of molecular evolution, an evolutionary algorithm for combinatorial optimization has been developed. This algorithm consists of a versatile variation scheme and an innovative decision rule, the essence of which lies in a radical revision of the conventional philosophy of optimization: A number of configurations of variables with better values, instead of only a single best configuration, are selected as starting points for the next iteration. As a result the search proceeds in parallel along a number of routes and is unlikely to get trapped in local optima. An important innovation of the algorithm is introduction of a constraint to let the starting points always keep a certain distance from each other so that the search is able to cover a larger region of space effectively. The main advantage of the algorithm is that it has more chances to find the global optimum and as many local optima as possible in a single run. This has been demonstrated in preliminary computational experiments.