Proteinoids acids

On a weight basis, thermal polylysine was three times as active as lysine-rich proteinoid. Acidic proteinoid showed little or no activity (Table X). Second-order rate constants were, respectively, 0.054, 0.012, and 0.0006 liter/gm per minute (these values being corrected for the spontaneous rate of 0.003 min i). Multiple samples of a particular kind of thermal polymer showed similar levels of activity ( 10 relative %). 

S. W. Fox (from 1984, director of the Institute for Molecular and Cellular Evolution of the University of Miami) made the highly controversial suggestion that the amino acid sequences in the proteinoids are not random. Nakashima prepared a thermal polymer from glutamic acid, glycine and tyrosine the analysis showed that two tyrosine-containing tripeptides had been formed pyr-Glu-Gly-Tyr and pyr-Glu-Tyr-Gly (Nakashima et al 1977). The result was confirmed (Hartmann, 1981). A closer examination of the reaction mechanism showed that the formation of these two tripeptides under the reaction conditions used depends on three parameters ... 

The literature of metabolism in proteinoids and proteinoid microspheres is reviewed and criticized from a biochemical and experimental point of view. Closely related literature is also reviewed in order to understand the function of proteinoids and proteinoid microspheres. Proteinoids or proteinoid microspheres have many activities. Esterolysis, decarboxylation, animation, deamination, and oxido-reduction are catabolic enzyme activities. The formation of ATP, peptides or oligonucleotides is synthetic enzyme activities. Additional activities are hormonal and inhibitory. Selective formation of peptides is an activity of nucleoproteinoid microspheres these are a model for ribosomes. Mechanisms of peptide and oligonucleotide syntheses from amino acids and nucleotide triphosphate by proteinoid microspheres are tentatively proposed as an integrative consequence of reviewing the literature. 

Proteinoids, as a model of primitive abiotic proteins >, are formed by polymerization from protobiologically plausible micromolecules (amino acids) under presumed protobiological conditions. Proteinoids have enzyme-like activities and metabolic qualities. Proteinoid microspheres are the most suitable model for protocells since they do not consist of macromolecules extracted from contemporary organisms. 

The proteinoid microspheres (Fig. 1), as simulated protocells, form from virtually all of the known wide variety of thermal copolyamino acids 2). Microspheres are formed if the aqueous or aqueous salt solution of proteinoid is heated and the clear decanted solution is allowed to cool. This self-assembly may also be effected by chilling solutions saturated at room temperature. Sonication at room temperature can be used. 

When the pH of a suspension of microspheres of acidic proteinoid is raised by 1-2 units, diffusion of material from the interior to the exterior, fission into two particles, and the appearance of a double layer in the boundary are observed 2 Proteinoid microspheres shrink or swell on transfer to hypertonic or to hypotonic solutions respectively. Some experiments show that polysaccharides are retained under conditions in which monosaccharides diffuse out2. Some proteinoid microspheres possess the intrinsic capacity to grow by accretion, to proliferate through budding, and to form junctions 2). The morphology and other characteristics of proteinoid microspheres are altered by the inclusion of other materials such as polynucleotides, lipids or salts. 

Acidic proteinoids accelerate the hydrolysis of the unnatural substrate, p-nitrophenyl acetate 7,8). P-Nitrophenyl acetate has been used as a substrate for both natural esterases and esterase models. The imidazole ring of histidine is involved in the active site of a variety of enzymes, including hydrolytic enzymes. Histidine residues of proteinoid play a key role in the hydrolysis, the contribution to activity of residues of lysine and arginine is minor, and no activity is observed for proteinoid containing no basic amino acid 7). 

The proteinoids are inactivated by heating in buffer solution or by treatment with alkali at room temperature, and it is proved that the hydrolysis of cyclic imide bonds, in which aspartic acid residues are initially bound, accompanies the inactivation by heat8). 

Alkali-treated proteinoids containing the 18 common amino acids promote the hydrolysis of the ester bond of p-nitrophenylphosphate 9). In general, the higher the proportion of neutral and basic amino acids proteinoid has, the higher the activity is9>. 

After the cooling of a hot solution of acidic proteinoid and zinc hydroxide, zinc-containing microspheres are deposited. The Zn-proteinoid microspheres washed with water retain the activity, but the wash liquids show successively less activity U). Attempts to introduce Zn into proteinoids in this manner gave highly erratic results until freshly prepared zinc hydroxide was used n). There have not been further experimental data published these experiments were mainly to attempt introducing zinc into proteinoid microspheres. 

Proteinoids catalyze the decarboxylation of oxaloacetic acid to pyruvic acid, lysine-rich proteinoid being the most effective of these tested. Some are about 15 times more active than the equivalent amount of free lysine. Acidic proteinoids exhibit very little activity18). 

Proteinoids were tested after being stored in the dry state for 5 to 10 years. Acidic proteinoids effective in catalyzing the hydrolysis of p-nitrophenyl acetate showed the same levels of activity as observed 10 years earlier19>. Lysine-rich proteinoids which catalyzed the decarboxylation of oxaloacetic acid were found to be insoluble in assay medium after 5 years of storage in the dry state. Their activity, however, had increased by 32 to 145%, and the activity of the lysine-rich proteinoids was largely associated with the insoluble portion 19). 

Proteinoids accelerate the conversion of pyruvic acid to acetic acid and carbon dioxide. In a typical experiment, a mixture of 20 mg of proteinoid and 0.4 mg of radioactive pyruvic acid in 20 ml of 0.2 NTris buffer, pH 8.3, is incubated at 37.5 °C for 1, 2 or 3 days. The evolved 14C02 is absorbed in NaOH solution, and acetic acid is identified by paper chromatography and by preparing a derivative of acetic acid, namely p-bromophenacyl acetate 20). 

In general, acidic proteinoids are more active than lysine-rich proteinoids for this reaction. Thermal poly(glutamic acid, threonine) and thermal Poly(glutamic acid, leucine) are the most active of these tested 20>. The activity is gradually decreased by progressive acid hydrolysis20. Compared with natural enzymes, the activity of proteinoid is weak. However the decarboxylation of pyruvic acid by proteinoid obeys Michaelis-Menten kinetics as expressed by the Lineweaver-Burk plot201. In this reaction a small amount of acetaldehyde and acetoin are formed in addition to acetic acid and C02 201. 

Light enhances decarboxylation activity by proteinoids, with pyruvic acid, glucuronic, acid, glyoxalic acid, citric acid or indole-3-acetic acid as substrates 22,23). In a typical experiment, 20 mg of proteinoid is incubated with 20 pmoles of radioactive substrate for 2-3 days in 10 ml of buffer pH 4.5 (or 7.0) at 37 °C, under the irradiation of a tungsten filament bulb with a filter of 10 % CuS04 solution the COa evolved is trapped as sodium carbonate 22). 

Thermal polylysine catalyzes the formation of glutamic acid from a-ketoglutaric add with urea and Cu2+ 24). A reaction mixture of CuS04, urea, a-ketoglutaric acid (0.1 mM each) and 10 mg of proteinoid in 6 ml of pH 7.0 buffer is incubated at 37.5 °C for 24 hours. The glutamic acid formed in the reaction mixture was identified on an amino acid analyzer after desalting on an ion-exchange column 24>. 

Thermal polylysine also catalyzes the formation of a-ketoglutaric acid from glutamic acid with CuCl25). A reaction mixture of lysine-rich proteinoid (20 mg), 14C(U)-l-glutamate (0.1 mM), and CuCl (0.1 mM) in 6 ml of Britton-Robinson buffer (pH 7.0) is incubated at 37.5 °C for 2 hours. More than 40 % of the radioactivity used is recorded in a-ketoglutaric add by paperchromatography of the reaction mixture25 . Free lysine and Leuehs poly-L-lysine have no activity 2S). The reaction obeys Michaelis-Menten kinetics at optimum pH 7.0 25). 

Hemoproteinoids have peroxidatic and catalatic activity 26). The peroxidase activity of hematin is increased up to 50 times when the hematin is incorporated into proteinoids 26), The hemoproteinoids have been synthesized from various mixtures of amino acids containing 0.25-2.0% hematin. The isoelectric point of the lysine rich hemoproteinoids is about 8 and the molecular weights are a little below 20,000 by gel filtration 26). 

A small part of metabolic pathways by proteinoids has been conceptualized2,5>. A flow from oxaloacetic acid to pyruvic acid 18 19) to acetic acid 20) and a side reaction from pyruvic acid to alanine, which is reversible are depicted in a conceptual integration of results2 5). 

Each of the reactions is catalyzed by a different type of proteinoid or metal-proteinoid complex. The reaction of pyruvic acid to alanine and the reverse reaction are hypothesized from the experimental results in the amination of a-ketoglutaric acid 241 and the deamination of glutamic acid 2S). 

Lysine-rich proteinoids in aqueous solution catalyze the formation of peptides from free amino acids, ATP (or pyrophosphate) and Mg2"1-. Figure 3 shows experiments in which diglycine and triglycine are thus produced 27). 

This catalytic activity is not found in acidic proteinoids nor in neutral proteinoids, even though they contain some basic amino acid, only in basic amino acid-rich proteinoids. No peptide is formed in controls containing free amino acids. The pH optimus for the synthesis is about 11, but is appreciable below 8 and above 13, the temperature data indicate an optimum at 20 °C or above, with little increase in rate to 60 °C 27). The yield from this experiment, based on glycine, is 0.40% for diglycine and 0.12% for triglycine 27). 

Fig. 4. Fractionation on Sephadex G-25 of the peptides of eighteen amino acids in the presence of histidine, lysine-rich proteinoid and ATP at pH 11 for 5 days Fox, Nakashima 291...

While the experiments of Fig. 3 were performed with glycine, other peptides have been synthesized from other amino acids such as lysine, phenylalanine or proline 27 28). Peptide synthesis from an eighteen amino acid mixture has been also demonstrated by using (histidine and lysine)-rich proteinoid. When the product is fractionated on Sephadex G-25, most of the oligopeptides appear to be in the dipeptide-tripeptide range or larger, and little of free amino acids survive from the reaction. Virtually all types of amino acid appear to yield peptides (Fig. 4) 29). 

The synthesis of peptide has been tested at pH 7.2 in a suspension of (acidic + basic) proteinoid microspheres. The activity of the complex particles is several times as large as that of the basic proteinoid solution alone 28). 

The syntheses of peptides are energized by ATP, they are operative in the presence of water, and the ATP is most effective in the peptide synthesis when supplied continuously. If the ATP is supplied in repeated small fractions, the conversion from amino acid to peptide in the reaction mixture with acidic and basic proteinoid microspheres is greater than if the same total amount is supplied at once, since the amino acid is present in considerable molar excess over ATP. The most likely explanation for the higher rate of conversion for repeated reaction of ATP is that it is more efficiently used, due to rapid decay of any one charge of ATP 28>,... 

It is remarkable that proteinoid microspheres bring the optimum pH for the amino acid activation from the acidic range to neutral. Acidic condition for the activation may be provided in the metal-proteinoid microspheres in neutral buffer. 

The mode of peptide synthesis by proteinoids or proteinoid microspheres has not so far been ascertained. We propose here possible ways to synthesize peptides from amino acid and ATP by proteinoids, as shown below. Since it is difficult to deduce mechanisms from the data reported, the following scheme is tentative. 

The intermediate for peptide synthesis is probably aminoacyl 5 -adenylate, formed from amino acids and proteinoid nucleotide complex. In the proteinoid nucleotide complex, the phosphate of adenylate may be attached to the imidazole of histidine in... 

Acidic proteinoid potentiates the active structure of lysine-rich proteinoid participating in forming microspheres in neutral buffer. Physical surface effects and providing micro condition in the microspheres could be surmised. Activation of amino acids generally requires acidic condition. Amino acids are activated by ATP and Mg2+ at pH 4-5 32 33). Aminoacyl adenylate anhydride and ester is formed preferentially from amino acid and adenylate imidazolide at pH 6.0J7). On the other hand, polycondensation of activated amino acids undergoes at pH values higher than 7. Peptides are formed from aminoacyl adenylate in basic buffer (the optimum pH is 10 for alanyl adenylate 40) from amino acid adenylate phosphoramidate and imidazole at pH 7.0 from N-(aminoacyl)-imidazole at pH 6-9 43). In this context, acidic and basic environments may be provided inside and/or on the surface of the microspheres composed of acidic and basic proteinoids in neutral buffer. Acidic micro condition suitable for the activation of amino acids and basic micro environment favorable for peptide formation from activated amino add may be provided. 

A mixture of 0.44 g of acidic proteinoid, 0.19 g of basic proteinoid, and 0.31 g of radioactive ATP in 2.0 ml of 0.02 M MgCl2, 0.05 M Tris buffer, is heated in a boiling water bath for 5 min, and then incubated at 37 °C for 24 hrs, after the fractionation of the mixture by DEAE-cellulose column chromatography. Oligonucleotides produced are identified with those of authentic markers 47). Chain length has been determined by the ratio of AMP to adenosine in the alkaline hydrolyzate of the material which was treated by alkaline phosphatase to remove 5 - and 3 -phosphate47). 

ATP is converted to dinucleotide in magnesium-containing buffer without proteinoid in a yield of 0.7 %, but in the presence of basic proteinoid solution, acidic proteinoid microspheres, or acidic-basic proteinoid microspheres, the conversion to oligonucleotides is 2.2-2.3 %. With AMP instead of ATP no oligonucleotides result. The proteinoids are thus a model for proto-RNA polymerase activity, and ATP is reaffirmed as the source of energy for the synthesis of phosphodiester linkages 47). The microspheres of acidic proteinoid or of acidic-basic proteinoid synthesize di- and trinucleotides, while without proteinoid or basic proteinoid solution dinucleotide only results. Furthermore, the ratios of trinucleotides/dinucleotide are 0.5 for acidic-basic proteinoid microspheres, 0.2 for acidic proteinoid microspheres 47). 

Experiments have revealed that the microsystems of proteinoid and polynucleotide have stability in solution under changing temperature of pH such as is not possessed by particles composed of acidic proteinoid alone 2,54), The nucleoproteinoid microparticles have been viewed as models of evolutionary precursor of ribosomes 54). 

Various lysine-rich proteinoids were tested for their ability to form microparticjes with yeast RNA. This ability was found to appear at a proportion of basic to dicarb-oxylic amino acid of approximately 1.054). Mixing of dilute solutions of lysine-rich proteinoid and of RNA yields small globular microparticles which are not dissolved by heating. On the other hand, when a sufficiently lysine-rich proteinoid is allowed to interact with calf thymus DNA, fibrous material results54). 

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