On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies

Steady-state kinetic study of the inhibition of 3-phosphoglycerate kinase reaction by the substrate analogues D-glycerol 3-phosphate, 2-phosphoglycolate, tartronate and malonate revealed competition with respect to 3-phosphoglycerate. D-Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3-phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3-phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl-lacking D-glycerol 3-phosphate is a non-competitive inhibitor with respect to MgATP, while all the investigated carboxyl-containing inhibitors compete for MgATP binding. The inhibitory analogues of 3-phosphoglycerate reduce the reactivity of both the two fast-reacting and the five slow-reacting thiol groups of the enzyme molecule. In the case of the fast-reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate-containing nucleotides and analogues slow down significantly the reaction rate of the fast-reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X-ray structural data. The fast-reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand-induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate-binding properties of carboxamidomethylated and the unmodified enzyme.     

Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase

Brzozowski, Thomas H. (Stanford University School of Medicine, Palo Alto, Calif.), and Sumner M. Kalman. Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase. J. Bacteriol. 91:2286-2290. 1966.-Earlier studies have shown that the carbamyl phosphate synthesis from ammonia in cell extracts of wild-type Escherichia coli is due to at least two enzymes, acetokinase and the glutamine-dependent carbamyl phosphate synthetase. Partial purification of the glutamine-dependent carbamyl phosphate synthetase and acetokinase fails to separate from these enzymes this ammonia-dependent activity. An antibody to the partially purified acetokinase was prepared and used to determine the distribution of the ammonia-dependent activity in wild-type organisms and single-step arginine-uracil-requiring mutants with respect to the two enzymes. Such a study was possible because the antibody inhibits acetokinase but not the glutamine-utilizing carbamyl phosphate synthetase. Enzyme inhibition obtained by the stepwise addition of the antibody to cell extracts indicates that all of the ammonia-dependent carbamyl phosphate synthesis observed in the arginine-uracil-requiring mutants is due to a protein in the acetokinase fraction, presumably acetokinase itself, since acetyl phosphate and carbamyl phosphate synthesis were inhibited in a parallel fashion. In wild-type organisms, there is only partial inhibition of the ammonia-dependent activity, even when enough antibody is added to produce maximal inhibition of acetokinase. It is suggested that this residue is due to the glutamine-dependent carbamyl phosphate synthetase, for the ratio of the antibody insensitive to antibody sensitive ammonia-dependent activity present in cell extracts of the two wild-type organisms reported is qualitatively proportional to the level of carbamyl phosphate synthetase present relative to acetokinase.     

A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.     

The developing visual system and metamorphosis in the lamprey

Metamorphosis of the sea lamprey, Petromyzon marinus, is a true metamorphosis. The larval lamprey is a filter-feeder who dwells in the silt of freshwater streams and the adult is an active predator found in large lakes or the sea. The transformation usually occurs in the fifth or sixth year of life. Enlargement of the eye has been long accepted as a distinctive indication of metamorphosis in the sea lamprey, but it had been thought that this was because eye development in the larva was arrested after the formation of only the small central region. Recent studies indicate that all of the retina begins its development in the larva and that ganglion, amacrine, and horizontal cells differentiate in the peripheral retina of the larva. Retinal development is arrested during the premetamorphic period, to be resumed during metamorphosis. Metamorphic contributions include the differentiation of photoreceptor and bipolar cells. With the early appearance of ganglion cells, retinal pathways to the thalamus and tectum are established in larvae, as is a centripetal pathway. Tectal development spans the larval period but a spurt in tectal growth and differentiation is correlated with the completion of the retinal circuitry late in metamorphosis. The metamorphic changes in retina and tectum complete the functional development of the visual system and provide for the adult lamprey's predatory and reproductive behavior.     

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.     

Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field.

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.