FITC-labeled lectins and calcofluor white ST as probes for the investigation of the molecular architecture of cell surfaces. Studies on conjugatophycean species

Summary In a screening program with 7 FITC-labeled lectins as probes, ConA receptors were identified in all of the 28 members of theConjugatophyceae, being under investigation. In nearly all of them RCA₁₂₀ receptors, too, are expressed. In 3 species only, PNA receptors, and in 2 species UEA receptors have been detected. No binding of DBA, SBA, and WGA was observed. The receptors for ConA, RCA₁₂₀, and UEA were shown to be associated with different molecules. Each lectin exhibits a unique and specific binding pattern, both chemically, as well as with regard to the topographic distribution on cell surfaces. While ConA receptors predominantly are associated with constituents of the cell wall, RCA₁₂₀ receptors mostly form part of the surrounding mucilage; the same holds for UEA receptors. Besides a variability of topographic distribution and species-to-species variation, a cell-to-cell variation exists in many species, suggesting that the expression of a lectin receptor is due to the developmental state of the cell and/or depends on external stimuli. In conclusion, we may point out, that FITC-labeled lectins turned out to be extremely useful probes for the investigation of the molecular architecture of cell walls. Calcofluor white ST binding to fibrillar polysaccharides (most probably cellulose) was shown to be inhibited by external incrustations of the cell wall. One species does not show any reaction with calcofluor white ST at all.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

The old REH theory remains unsatisfactory and the new REH theory is problematical - a reply to holmquist and jukes

Summary In response to criticism of REH theory (Fitch 1980), Holmquist and Jukes (1981) have mostly avoided the criticism or misunderstood it. Since they themselves state in their response that Amino acid sequence data alone cannot be used to estimate total nucleotide substitutions, they agree with the criticism. Most of their paper treats the newer theory (here designated as the REHN theory) which attempts to use the nucleotide sequences encoding proteins to better estimate total nucleotide substitutions (Holmquist and Pearl 1980). Since I made no criticism of REHN theory, their comments are frequently beside the point of my original criticism of REH theory. Nevertheless, it is shown here that REHN theory is also unsatisfactory in that: One, the varions are now more clearly defined but in such a way as to preclude the same codon from suffering a nucleotide substitution in more than one evolutionary interval. Two, the set of codons that accepts silent substitutions is identical to the set that accepts amino acid changing nucleotide substitutions. Three, the uncertainty in the REH estimate is considerable in that alternative excellent fits to the same observatuonal data may give alternative REH values that differ significantly even before stochastic variation and selective bias are considered. Four, the fit of their model to data is an irrelevancy where there are zero degrees of freedom.     

An exceptional amino acid replacement on the distal side of the iron atom in proboscidean myoglobin

Summary Amino acid sequence determination of elephant myoglobin revealed the presence of the unusual substitution E7 His Gln. Stereochemical analyses suggest that the most suitable residue which can functionally substitute for His at this position in vertebrate globins is Gln. Physiochemical studies imply that the slower rate of autooxidation of elephant myoglobin is the result of this substitution which may confer some selective advantage on the species. Comparative sequence data of paenungulate myoglobins suggest that the His Gln mutation probably occurred in an ancestor of Elephantinae.     

Effects of Acute and Chronic Denervation on Release of Acetylcholinesterase and Its Molecular Forms in Rat Diaphragms

Abstract: Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE; EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3-h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amount of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, corresponded to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragm.     

Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

Evolutionary processes and evolutionary noise at the molecular level

Summary On account, notably, of a competition between different component functions for individual sites in polypeptide chains, each protein molecule represents a functional compromise, with some functions optimized, but the overall state of the molecule –suboptimal–. The proposal is made that the selection coefficient relating to a protein molecule under given conditions can in principle be broken down into partial selection coefficients relevant to the different functions that the molecule carries out. At generalfunction sites, each fixation improves some function, while others deteriorate, at first nonsignificantly, and the overall adaptive state of the molecule fluctuates around its maximum. A selective mechanism is described whereby kaleidoscopic changes in primary structure at variable sites are indefinitely promoted, independently of any environmental changes and with the molecule remaining close to a state of maximal overall adaptation. The paradoxical aspect of this proposal is analyzed. The implication of specific functions in substitutions at general-function sites is noted. Further, it is shown that a certain category of changes in the internal environment of the organism can be integrated into the constantenvironment model for selection. Genetic sufficiency is considered a notion more adequate than genetic optimality for describing biological fitness and for providing a basis for the present model. On this basis selection occurs without genetic load. Multipolymorphism is one of the consequences. Several lines of evidence, in particular observations on polymorphism in deep sea organisms, seem to support the model. It is pointed out that it provides a theoretical foundation for a molecular evolutionary clock. The theoretical constancy of the clock depends on the constancy of functional density. The question of the evolution of functional density is examined. Comparisons of observed substitution frequencies with values expected on a random basis are rejected as a measure of the contribution to evolution of nondetermination. They are considered to reflect a hierarchy in the resistance of the molecules to different amino acid residues as substituents. A limited component of –true– randomness, again accompanied by selection, is on the other hand provided by the model. Most amino acid substitutions are considered evolutionary noise, even though noise compatible with selection. It is proposed that evolutionarily significant substitutions may be identified by monitoring changes in functional density and weighted functional density.Directeur de Recherche at Centre National de la Recherche Scientifique, Paris.