The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.     

The determination of the helical screw angle of a helical particle from its diffraction pattern

A Fourier analysis of the distributions of different types of amino acids in the sequence of tropomyosin shows strong 14th-order peaks in the profiles of both negatively charged and non-polar amino acids, with a period of $\text{19}\text{2}\text{3}$ residues and an overall repeat length of 275 ± 2 amino acids, which is shorter than the sequence length of 284 amino acids. Both peaks are statistically significant and confirm Parry's work (1974, 1975b). The regularities are analysed in terms of an assumed supercoil structure in which two α-helices lie parallel and in register to form a supercoil with a pitch of 137 Å. These molecules are then assumed to overlap end-to-end by eight to nine amino acids so that the periodicity is continuous along an extended filament of linked tropomyosin molecules. The periodic features are stronger in the outer surface of the molecule away from the core of the supercoil. The sequence divides into 14 bands which each have a narrow zone of net positive charge and a broader negatively charged zone. Overlapping every positive zone is a hydrophobic zone which always has at least one non-polar group on the outer surface. Anomalies in the charge distribution are found near the molecular ends and close to Cys190. These are attributed to the end-to-end overlap site and the troponin binding site.In the thin filament the 137 Å pitch supercoil would make seven half-twists relative to the twisted actin helix along a 385 Å length, so that a pair of adjacent bands would be oriented equivalently with respect to a pair of actins 28 Å apart. We therefore suggest that the bands (each containing one zone of each type) should be divided alternately into two series, α and β. Every pair of bands is $\text{39}\text{1}\text{3}$ residues long and each of the seven pairs corresponds with one segment of the 42-residue gene duplication repeat observed previously in the sequence. The disparity between the periods of 42 and $\text{39}\text{1}\text{3}$ is overcome by deletions and insertions. The $\text{39}\text{1}\text{3}$-residue periodicity is not simply a consequence of the supercoil structure or gene duplication but is probably a result of adaptation to the spatial periodicity of the actin helix in muscle. Although the α and β bands are alike in general, they differ systematically in detail and the α bands are more regular than the β.We propose that the seven α and seven β bands are alternative sets of sites which bind equivalently to complementary groups of sites on seven actins in the “relaxed” and “active” states of muscle, respectively. In each band the negative zone probably attaches to actin by magnesium bridges and the hydrophobic zone by direct contacts with the narrow outer edge of the supercoil. Since the supercoil twists 90 ° relative to actin on passing between adjacent α and β bands, a quarter rotation of the whole tropomyosin molecule would detach one set of seven sites and attach the other, allowing a highly co-operative switch mechanism.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Comparative kinetic-ionic strength study of two differently charged cytochrome C: effects are limited to overall charge.

The reduction kinetics of two differently charged cytochromes $\text{c}$, horse cytochrome $\text{c}$ and $\text{Rhodosprillum rubrum}$ cytochrome $\text{c}{}_2$, by ferrous EDTA^2? were studied as a function of ionic strength. Since both proteins have nearly the same heme edge region, but have very different overall surface charge, this comparative study served as a direct test of the utility of small nonbinding non-physiological redox agents in the study of the charge of electron transfer sites of redox proteins. Calculations based on the ionic strength-kinetic data yielded protein charges of +10 and +2.3 for cytochrome $\text{c}$ and cytochrome $\text{c}{}_2$ respectively and compared well with values of +9 and +3 for the overall charge of the proteins based on acidic and basic amino acid residues. It is concluded that ionic strength effects upon the redox kinetics with such nonbinding nonphysiological redox agents reflect the influence of the overall protein charge and not the localized charge of the presumed site of electron transfer.     

Effect of phenethylbiguanide on sugar and amino acid transport in rabbit ileum

Phenethylbiguanide has been shown to be an inhibitor of sugar and amino acid uptake in both $\text{in vivo}$ and $\text{in vitro}$ conditions. This action could be due to a competition for sodium sites on the sugar and amino acid carrier molecules. The effects of phenethylbiguanide on $\text{in vitro}$ intestinal preparations indicate that this compound has a time-dependent effect, it is most effective when placed on the mucosal surface but is also effective on the serosal surface. Furthermore, competition studies indicate that it is a competitive inhibitor of sugar uptake and a non-competitive inhibitor of amino acid uptake. These results are consistent with the differences in the mechanism of coupled transport between sugars and amino acids, but, do not substantiate the idea that phenethylbiguanide competes for the sodium site on the ternary carrier.     

Steady-state current-voltage characteristics of amino acid transport in rhizoid cells of Riccia fluitans Is the carrier negatively charged?

The amino acid transport across the plasmalemma of Riccia fluitans rhizoid cells has been further characterized by means of current-voltage $\text{I?V}$) analysis. On the basis of two cyclic transport models which include six different carrier states, the question is raised, whether the electrochemical pH-gradient drives a negatively charged carrier or a positively charged alanine-proton-carrier complex across the membrane. $\text{I?V}$ analysis shows that (1) the typical $\text{I?V}$ characteristic of l-alanine transport follows a sigmoid curve, (2) maximal accumulation of l-alanine within the cytoplasm is reached after about 1 hour, (3) the electrically accessible cytoplasmic l-alanine concentration is limited to about 20 mM, and (4) the steady-state saturation current depends directly on external l-alanine concentration. It is concluded that (a) these results are consistent with the predictions of the models for a negatively charged carrier, and (b) that the rate-limiting step involves the translocation of the ternary complex.     

The effect of neutral and charged micelles on the acid-base dissociation of the local anesthetic tetracaine

The influence of surfactant micelles on the acid-base dissociation of the charged tertiary amino group of the local anesthetic, tetracaine, has been investigated. From measurements of tetracaine fluorescence as a function of bulk pH, apparent $\text{p}\text{K}$ values of 6.88, 7.58 and 9.92 were found in the presence of cationic, neutral and anionic micelles, respectively, in 10 mM NaCl. These values are considerably displaced with respect to the $\text{p}\text{K}$ in aqueous solution which is 8.26. Such large shifts can be attributed to the effect of the surface polarity and electrical potential on the dissociation behavior of the anesthetic bound to micelles. It can be expected that the acid-base dissociation of a local anesthetic adsorbed to nerve fibers will also be affected by the properties of the membrane surface. Thus, it is suggested that the influence of the interfacial region on the $\text{p}\text{K}$ of surface-bound molecules should not be disregarded when estimating the proportion of charged and uncharged forms of local anesthetics interacting with axonal membranes.     

Localization of translation initiation in the message for E. coli UDP-galactose 4-epimerase: the amino terminal sequence

The amino acid sequence of $\text{E.}$$\text{coli}$ UDP-galactose 4-epimerase has been determined through the amino-terminal 28-amino acid residues using an automated protein sequenator. Alignment of UDP-galactose operon messenger RNA and the amino acid sequence of epimerase demonstrates that the first 26 bases in the mRNA are transcribed but do not take part in translation of epimerase.     

Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P₁′,³ the residue furnishing the amino group to the scissile bond, produced significant changes in k_rmcat, whereas the identity of P₁′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P₁′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P₁t$\text{,}\text{?}$ P₂′, P₃′) are involved while only two residues (P₂, P₁) on the amino terminal side of the sensitive bond are implicated.     

Increase in initiation sites for chromatin directed RNA synthesis by acetylation of chromosomal proteins.

Treatment of rat liver chromatin with 0.7 mM acetic anhydride (1) leads to an approximately twofold increase in initiation sites for DNA-dependent RNA polymerase from $\text{E.}\text{coli}$. With reconstituted chromatin, in which only the histone moiety was acetylated, again a twofold increase in initiation sites could be observed, compared to control chromatin which had undergone the dissociation and reassociation procedure, but which had not been exposed to acetic anhydride.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Specific 125I-somatomedin receptor on circulating human mononuclear cells

Somatomedin is a growth hormone-stimulated peptide purified from plasma which has $\text{in vitro}$ actions on cartilage and other tissues. Specific receptor sites for somatomedin distinct from insulin receptor sites have been described for many tissues. This study demonstrates the existence of a specific ¹²⁵I-somatomedin receptor site on circulating human mononuclear cells. This will provide an easily accessible human source for the study of changes in somatomedin receptors in disease states.     

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes.

The rapid transport of $\text{L}$-cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the $\text{ASC}$ system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na⁺-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems $\text{A}$ and $\text{ASC}$, showing better accommodation of its two α-methyl groups by $\text{ASC}$ than in the Ehrlich cell. The hepatocyte $\text{ASC}$ system tolerates Li⁺-for-Na⁺ substitution better than does System $\text{A}$, although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System $\text{A}$.     

An Artemia salina factor which counteracts the mRNA-induced inhibition of initiator Met-tRNA binding to initiation factor eIF-2.

Ternary complex formation between eukaryotic initiation factor 2 (eIF-2), initiator Met-tRNA and guanosine 5′-(β, γ-imino) triphosphate [GMP-P(NH)P] is strongly inhibited by mRNA in the $\text{Artemia salina}$ system. Developing $\text{A. salina}$ embryos contain a factor which displays a novel activity, namely the ability to counteract the mRNA-induced inhibition of ternary complex formation. This factor is heat-labile. It is proposed that the factor may play an important role in protein biosynthesis by preventing mRNA from inhibiting an early step of peptide chain initiation.     

Carbodiimide-dependent inactivation of dihydrofolate reductase

Dihydrofolate reductase from amethopterin-resistant $\text{Lactobacillus}\text{casei}$ was inactivated by a water soluble carbodiimide, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HCl. The rapid inactivation observed at pH 5.0–6.0, coupled with lack of recovery of activity from inactivated samples incubated with NH₂OH was consistent with modification of enzymic carboxyl groups. Significant protection against inactivation was provided by 7,8-dihydrofolate and NADPH. Analysis of the reaction order suggests that the carbodiimide-dependent inactivation may result from modification of a single essential carboxyl group.     

Effects of 6-thioguanine-containing RNA on in vitro protein synthesis

6-Thioguanine was administered to rats 12 hr after partial hepatectomy at a dose of 40 mg/kg of body weight; 6 hr later, polyadenylic acid-containing RNA was isolated and was used to measure initiation of protein synthesis $\text{in vitro}$ in a wheat germ system. $\text{In vitro}$ initiation was found to be 2.3-fold greater when 6-thioguanine-containing RNA was employed, than when polyadenylic acid-containing RNA isolated from untreated animals was used. The homopolymer, poly(TG), did not promote peptide synthesis in the wheat germ $\text{in vitro}$ system employed.