Thermodynamics and kinetics of co-operative protein-nucleic acid binding: I. General aspects of analysis of data

The process under consideration is the binding of a ligand to a linear polymer of equivalent subunits such that each bound molecule of ligand occupies n subunits. Interactions between bound ligand molecules are also considered. Some useful points regarding the evaluation of raw data without recourse to any specific binding mechanism are discussed first. For a treatment in terms of appropriate thermodynamic parameters a simple model is examined in greater detail. It assumes that interactions are limited to those between ligands bound to nearest-neighbour positions on the polymer.Exact expressions for some basic binding properties of this model at equilibrium are developed. The relations can be considerably simplified in the case of pronounced positive co-operativity which is frequently encountered in practice. Appropriate plots of the data to test the model and to evaluate its parameters are proposed.A simple but consistent kinetic scheme is also introduced. It allows calculation of relaxation times as they can be measured by means of special techniques.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of N^α-[¹⁴C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of N^α-[¹⁴C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 10⁷ to 2.6 × 10⁸m^?1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.     

Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung

Mouse Ehrlich ascites tumor cells incubated with the creatine analog, N-ethylguanidinoacetate (N-amidino-N-ethylglycine), accumulated up to 8 μmol/g packed cells of the creatine-P analog, N-ethylguanidinoacetate-P. Evaluation of N-ethylguanidinoacetate-P as a synthetic phosphagen under in vivo conditions was performed with Ehrlich cells loaded with equimolar amounts of a common reference phosphagen, cyclocreatine-P (1-carboxymethyl-2-imino-3-phosphonoimidazolidine) plus either N-ethylguanidinoacetate-P or creatine-P. It was concluded that N-ethylguanidinoacetate-P has a Gibbs free energy of hydrolysis equal to that of creatine-P and 2 kcal/mol greater than that of cyclocreatine-P. The relative rates of utilization of intracellular phosphagens by Ehrlich cells when their ATP pools were depleted with 2-deoxyglucose were in the order: creatine-P > N-ethylguanidinoacetate-P > cyclocreatine-P. Dietary N-ethylguanidinoacetate was nontoxic even at very high levels to all animal systems tested. Feeding of 2% N-ethylguanidinoacetate to mice or chicks resulted in equimolar replacement of natural by synthetic phosphagen to the following extents: heart, 75%; leg muscle, 50%; and brain 10–25%. N-Ethylguanidinoacetate-P is the most active synthetic phosphagen thus far found to be accumulated by animal tissues.     

Synthesis of specific membrane proteins is a function of DNA replication and phospholipid synthesis in Caulobacter crescentus

Analysis of the effects on membrane function and protein composition of altering phospholipid synthesis in Caulobacter crescentus showed that, like other bacteria, C. crescentus continues to induce a lactose transport system and to synthesize most membrane proteins. However, we show that the incorporation of a set of outer membrane proteins primarily synthesized in stalked cells is dependent on DNA replication which, in turn, is dependent on membrane phospholipid synthesis. Furthermore, the incorporation of another set of membrane proteins, two of which are synthesized primarily in the swarmer cell, appears to be independent of the replication of the chromosome but to be directly dependent on phospholipid synthesis. We have also found that when phospholipid synthesis is blocked, the synthesis of the flagellar proteins is inhibited and that this effect may be mediated by the primary inhibition of DNA replication. Newton has presented evidence that the synthesis of flagellar proteins is dependent on specific execution points in DNA replication and that this connection serves as a temporal regulator of differential protein synthesis (Osley et al., 1977; Sheffery & Newton, 1981). We suggest here that a direct link between the replicating chromosome and the growing membrane might serve, in turn, to dictate the site of membrane assembly of newly synthesized gene products.     

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol

A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH₄) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.     

6-substituted purines: a novel class of inhibitors of endogenous protein degradation in isolated rat hepatocytes

About 100 different purine derivatives and analogs were tested for their effect on protein synthesis and protein degradation in isolated rat hepatocytes. These included 6-aminopurines (adenine and adenosine analogs), 6-mercaptopurines, chloropurines, oxypurines, cytokinins, methylxanthines, methylindoles, benzimidazoles, and benzodiazepines. Most of the compounds were either inactive or inhibited protein synthesis as much as or more than they inhibited protein degradation. However, three methylated 6-aminopurines (3-methyladenine, 6-dimethylaminopurine riboside, and puromycin aminonucleoside) and four 6-mercaptopurines (6-methylmercaptopurine, 6-methylmercaptopurine riboside, 6-mercaptopurine riboside, and 2′,3′,5t$\text{-}\text{?}$triacetyl-6-mercaptopurine riboside) had a markedly stronger effect on protein degradation than on synthesis, and might therefore be potentially useful as selective degradation inhibitors. None of the seven above-mentioned purines had any significant effect on the degradation of the exogenous protein, asialofetuin, and would therefore seem to selectively inhibit endogenous protein degradation. Since the degradation was not further affected by purines in the presence of amino acids or lysosomotropic amines, it is suggested that the purines exert their effect specifically upon the autophagic/lysosomal pathway. All the mercaptopurines significantly depressed cellular ATP levels, whereas the methylated aminopurines did not. For this reason, the latter are probably more useful as degradation inhibitors. 3-Methyladenine had no effect on protein synthesis at a concentration (5 mm) which inhibited protein degradation by more than 60%, and may therefore be regarded as a highly specific inhibitor of autophagy.     

Three-dimensional structure of a cell wall glycoprotein

Electron microscopy and computer image analysis have been used to determine the three-dimensional structure of the crystalline glycoprotein cell wall layer of the alga Lobomonas piriformis. Images of negatively stained specimens, tilted through a range of angles up to 70 °, were combined to give a map of the molecular envelope to a resolution of 2.0 nm. The cell wall layer consists of crystalline plates the centres and edges of which display distinctly different but isomorphous structures. A comparison of three-dimensional reconstructions of the two areas shows the difference probably to be due to a conformational change of one of the glycoprotein subunits. The structure consists of two sets of dimers composed of rod-shaped subunits which lie with their long axes approximately in the plane of the crystal. The centre-edge transition may have significance in the pathway of accretion of new subunits during cell wall growth.     

Properties of a 5'-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 mm, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 mm were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg²⁺, Mn²⁺, Ca²⁺, Fe²⁺, or Zn²⁺ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.     

Caulobacter crescentus flagellar filament has a right-handed helical form

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.     

How does limited trypsinization influence the surface structure and binding of calcium to lipoprotein (a): a spin-labeling study

Enzyme kinetic studies are presented which demonstrate the activating effect of phosphate on the conversion of pyruvate to lactate by rabbit muscle lactate dehydrogenase. A simple method of active enzyme gel chromatography is used to preclude the possibility that this effect is due to redistribution of enzyme between tetrameric and dissociated states as the result of preferential binding of phosphate to the tetrameric enzymatic form. By analysis of the kinetic results in terms of an ordered two-substrate mechanism, the source of the activation is traced to enhancement of the strength of the enzyme-NADH interaction, primarily because of an increase in the rate constant for the formation of the binary enzyme-coenzyme complex. Preliminary estimates of the relevant equilibrium constants from the kinetic data indicate that the binding of phosphate to rabbit muscle lactate dehydrogenase leads to a two- to fourfold increase in the intrinsic association constant for the interaction between NADH and the enzyme under the conditions (pH 7.4, I = 0.15) used to study the activation phenomenon.     

Activation of a silent gene is accompanied by its demethylation

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.     

Photoaffinity labeling of a microtubule-associated cyclic AMP-binding protein

8-Azido cyclic AMP has been used as a photoaffinity probe to identify cyclic AMP-binding proteins in microtubule preparations. Bovine brain microtubule proteins and rabbit muscle protein kinase were incubated with the photoaffinity ligand in reduced light for 15 min, without additions or with 100-fold excess unlabeled cyclic AMP or 5′-AMP. Samples were then irradiated at 254 nm at a distance of 1 cm for 5 min, in ice. After irradiation aliquots were taken for electrophoresis in one or two dimensions. Polypeptides which bound the photoaffinity label were visualized by autoradiography. The apparent molecular weights of the most prominent 8-azido ³²P-cyclic AMP-binding proteins are in the same range as those of the RII of the muscle enzyme. Following two-dimensional electrophoresis the major microtubule-associated cyclic AMP-binding proteins resolve as two spots with about the same pI (~pH 5.0) but slightly different molecular weights. Both spots are in the molecular weight range of the tubulins but they are clearly resolved from the tubulins in the first dimension. Cyclic AMP, but not 5′-AMP blocks the labeling of these proteins. There are low levels of labeling of the tubulins, the high-molecular-weight MAPs and several polypeptides with molecular weights near tubulin but with more basic pI. The photoaffinity probe has demonstrated that the major microtubule-associated cyclic AMP-binding protein of bovine brain is distinct from other RII proteins and from tubulin isomorphs.     

Binding of terbium to apoferritin: a fluorescence study

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.     

Isolation of a multigene family containing human alpha-tubulin sequences

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 base-pairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A · T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions.The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.     

Asymmetric maturation of a dimeric transfer RNA precursor

Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNA^Ile is present in ≤ 30% the amount of tRNA^Thr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNA^Thr and tRNA^Ile, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNA^{Thr + Ile} is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNA^Ile can be explained by a novel processing pathway that generates a metabolically unstable tRNA^Ile product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNA^Ile has a unique physiological role.     

The synthesis of 8-(6-aminohexyl)-amino-GMP and its applications as a general ligand in affinity chromatography.

The steady-state kinetic behaviors of the five rabbit adrenal norepinephrine N-methyl transferase isozymes have been compared with particular reference to substrate inhibition patterns. Four distinct substrate inhibition patterns were observed. The E-1 isozyme was not subject to inhibition by either substrate, while the E-2 isozyme was inhibited by both substrates. The E-3 and E-4 isozymes were inhibited by norepinephrine only, while E-5 is inhibited only by S-adenosylmethionine. The substrate inhibition constants were sufficiently small in relation to the Michaelis constants to make substrate inhibition an important factor in regulation of activities of the isozymes.