Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range

The effects of nonsaturating amounts (5–60 nmol/mg membrane protein) of $\text{p-}\text{chloromercuribenzoate}$ on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher $\text{p-}\text{chloromercuribenzoate}$ concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled $\text{p-}\text{chloromercuribenzoate}$. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to $\text{p-}\text{chloromercuribenzoate}$. Other reagents including $\text{N-}\text{ethylmaleimide}$, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.     

The biosynthesis of ubiquitin by parathyroid gland

Data are presented on the isolation, biosynthesis, and identification of a small peptide (H) from parathyroid gland. Under our experimental conditions this peptide (H) represents, in addition to secretory protein-I and proparathyroid hormone, the other major protein which is rapidly synthesized during shorterm incubations of tissue slices. N-terminal sequence analysis was performed on samples of peptide H and the resulting data used to conduct a search of the sequence data bank. The search established the identity of peptide H as ubiquitin. These findings establish parathyroid gland as another system which rapidly produces ubiquitin $\text{in}$$\text{vitro}$, in addition to the systems employing hypothalamus and pituitary where ubiquitin biosynthesis was initially observed by Seidah $\text{et}$$\text{al}$ and Scherrer $\text{et}$$\text{al}$.     

Enantiomer selection in the nucleophilic addition reaction of optically active amines to α-amino acid N-carboxyanhydrides

The enantiomer selection in the nucleophilic addition reaction of optically active amines such as α-amino acid esters to phenylalanine and $\text{N-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$ in $\text{m-}\text{dimethoxybenzene}$ as a solvent has been investigated. Stereoselectivity between the amines and the $\text{N-}\text{carboxyanhydrides}$ was found to change markedly according to the reaction conditions. This experimental finding is in contrast to the idea hitherto accepted that in the nucleophilic addition-type polymerization of α-amino acid $\text{N-}\text{carboxyanhydride}$ the growing chain end reacts preferentially with one of the enantiomorphic $\text{N-}\text{carboxyanhydrides}$ having the same configuration, and indicates the importance of the investigation of stereoselectivity in the $\text{N-}\text{carboxyanhydride}$ polymerization using suitable model reactions. Most $\text{(S)-α-}\text{amino}$ acid esters reacted preferentially with $\text{(R)-}\text{phenylalanine}\text{N-}\text{carboxyanhydride}$, and this type of stereoselectivity increased with the $\text{N-}\text{methylation}$ of $\text{N-}\text{carboxyanhydride}$ and with increasing bulkiness of the C^α substituent of α-amino acid esters (alanine < norleucine < leucine < valine). The relationship observed between the stereoselectivity and the structures of amines and $\text{N-}\text{carboxyanhydrides}$ was explained satisfactorily in terms of the transition state model in which the interaction of $\text{N-}\text{carboxyanhydride}$ nitrogen and α-amino acid ester carbonyl as well as the interaction of $\text{N-}\text{carboxyanhydride}$ carbonyl and α-amino acid ester nitrogen was taken into account. $\text{(S)-}\text{Proline}$ ethyl ester did not show enantiomer selectivity toward phenylalanine $\text{N-}\text{carboxyanhydride}$, but reacted preferentially with $\text{(S)-(N)-}\text{methylphenylalanine}\text{N-}\text{carboxyanhydride}$. for the reaction of proline ester with $\text{N-}\text{carboxyanhydride}$ a transition-state model was proposed, which was different from the transition state model proposed for other α-amino acid esters. Some experiments were carried out to examine the transition-state models proposed. The implications of the present investigation in stereoselectivity in the nucleophilic addition-type polymerization of $\text{N-}\text{carboxyanhydride}$ hitherto reported are discussed.     

Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo.

Previous reports indicate that alterations of activity of cholinergic neurons $\text{in vivo}$ are followed by parallel changes in sodium-dependent high affinity choline uptake $\text{in vitro}$. These results are consistent with the proposal that this portion of choline uptake is regulatory in the synthesis of ACh. These results also suggest the possibility of utilizing sodium-dependent high affinity choline uptake as a measure of the relative state of cholinergic activity $\text{in vivo}$. In this study, we administer a number of drugs reported to alter turnover and release of ACh (both are measures of cholinergic activity $\text{in vivo}$, and subsequently examine sodium-dependent high affinity choline uptake $\text{in vitro}$. Administration of pentobarbital, chloral hydrate, morphine, physostigmine, Δ⁹ THC, hemicholinium-3 and oxotremorine, drugs which decrease ACh turnover and release, caused a reduction in choline uptake. Conversely, administration of pentylenetetrazol, atropine, scopolamine, and haloperidol, drugs which increase ACh turnover and release, caused an increase in choline uptake $\text{in vitro}$. These findings support the proposal that sodium-dependent high affinity choline uptake can be used as a relative measure of the activity of cholinergic neurons $\text{in vivo}$.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

ADP and ATP transport in mitochondria evidence for a metal-ion involved in transport catalysis by use of metallochromic indicators

This study introduces a new class of active-site directed probes with respect to ADP and ATP transport catalysis in rat liver mitochondria. The anionic monoazo dyes, e.g., $\text{p-(2-}\text{hydroxy}\text{-1-}\text{naphthylazo}\text{)}\text{naphtholsulfonic acid}$, are competitive inhibitors of carrier-mediated ADP uptake ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ 20–30 μM). The azo dyes also can displace the same amount of carrier-specific bound ADP as does carboxyatractyloside. Two essential substructures could be derived from a structure-activity study. Firstly, a sulfonic acid group in the para position relative to the azo bridge which becomes neutralized upon binding by a specifically located positive charge of the carrier protein. This electrostatic binding component, which presumably is represented by a strategic arginyl residue, seems to be essential for substrate binding as well as inhibitor binding. The second structural requirement for effective inhibition was found to be the $\text{o-}\text{hydroxy}$ or $\text{o,o′-}\text{dihydroxyazo}$ system, which is known to form stable complexes with metal ions by chelation. Experiments on prevention and reversal of dye-mediated inhibition revealed that the metal-chelating properties are responsible for the effects observed. In addition, using bovine serum albumin or the synthetic polymer Kollidone, inhibition could be prevented as well as abolished. It is postulated that a metal ion, possibly $\text{Mg}{}^{\mathrm{2+}}$, which is bound to the carrier protein plays an essential role for transport catalysis. The metal ion is assumed to form a functional ternary complex, i.e., a metal bridge complex between the carrier protein and its substrate.     

1-Aminoglycosides, a new class of specific inhibitors of glycosidases

1-Aminoglycosides represent a new class of specific and relatively potent inhibitors of glycosidases. These compounds are specific against those enzymes which act upon glycosides that correspond to glycone of the inhibitor. Thus α- and β-$\text{D}\text{=}$- are inhibited by $\text{D}\text{=}$-glucosidases but not by $\text{D}\text{=}$-galactosylamine and $\text{D}\text{=}$-mannosylamine. α- and $\text{D}\text{=}$-galactosidases are inhibited by $\text{D}\text{=}$-galactosylamine but not by the other two glycosylamines. $\text{D}\text{=}$-Mannosylamine inhibits mannosidase.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

Interaction of tritiated beta-endorphin with rat brain membranes

The potential usefulness of disaturated lecithin titer in plasma as an indicator of the presence of atherosclerosis has led to the development of a quantitative analytical method for the $\text{16:0}\text{16:0}$, $\text{16:0}\text{18:0}$, and $\text{18:0}\text{18:0}$ lecithins in plasma. The method employs extensive sample preparation to form diglyceride acetate derivatives, which are then separated by capillary column gas-liquid chromatography. The precision of the method is ±5% or better. Suggestions for large sample population analyses are included.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Metabolites of mating pheromone,rhodotorucine A, by a cells of Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ which induces mating tube formation of $\text{a}$ cells in $\text{Rhodosporidium}\text{toruloides}$ is metabolized rapidly by $\text{a}$ cells. By use of labeled rhodotorucine $\text{A}$, the degradation was found to be proteolytic. Two peptide fragments Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg and Asn-Gly-Cys(S-farnesyl) were identified as the metabolites. Proteolysis of the pheromone mainly occurred on the cell surface. Culture filtrate of $\text{a}$ cells at log phase did not metabolize rhodotorucine $\text{A}$.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

III. Possible mechanisms involved in the presynaptic cholinotoxicity due to ethylcholine aziridinium (AF64A) invivo

AF64A is a toxin which can diminish irreversibly cholinergic transmission $\text{in}$$\text{vivo}$ (1, 2). Disruption of neurotransmitter function $\text{in}$$\text{vivo}$ is specific to the cholinergic system when AF64A is administered in nanomolar quantities (3, 4). The mechanisms involved appear to be mediated presynaptically (2). The neurochemical and behavioral consequences of AF64A administration are reminiscent of similar measures in patients with Alzheimer's disease (5,6). Consequently, we have suggested tentatively that the AF64A treated animal may be explored as a potential animal model of this debilitating disease state (7). In this report we provide a brief overview of our recent findings using this compound $\text{in}$$\text{vivo}$, attempt to correlate these findings with those of others with similar aziridinium agents $\text{in}$$\text{vitro}$, and propose a possible mechanism of action of AF64A $\text{in}$$\text{vivo}$, based on recent observations made in our laboratories.     

An electron cytochemical study of 6-phosphogluconate dehydrogenase activity in infected erythrocytes during malaria

The distribution of 6-phosphogluconate dehydrogenase (6-PGD) was examined by an electron microscopic technique in erythrocytes of rodents infected with $\text{Plasmodium berghei}$, chickens with $\text{P. gallinaceum}$ and rhesus monkeys with $\text{P. knowlesi}$. Unlike glucose-6-phosphate dehydrogenase, which is restricted to the host erythrocyte, 6-PGD was found to be present in the parasite as well as the host erythrocyte in all infections studied. The implications of these findings are discussed in relation to the metabolism of malaria parasites.     

Controls of citrate synthase activity

The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an $\text{in vitro}$ catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration $\text{in vivo}$ compared to its concentration $\text{in vitro}$. In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available $\text{in vitro}$ so that little can be said concerning the $\text{in situ}$ control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes $\text{in vitro}$ can only serve as a guideline for parameters to study when techniques are available to study control of enzymes $\text{in situ}$.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

X-ray diffraction analysis of dehydrated myelin

Improved X-ray diffraction data from dry nerve myelin are presented. In addition to the spacings of approx. 150 Å, 60 Å, 44 Å and 34.6 Å, which have been previously reported, we identify a 14 Å series. The data suggests that the hydrocarbon chains in the single bilayer ($\text{≈ 60}\text{A}\text{?}$) is ordered, whereas in the double bilayer ($\text{≈ 150}\text{A}\text{?}$) and in the fluid phase ($\text{≈ 44}\text{A}\text{?}$) it is disordered. It is shown that cholesterol ($\text{≈34.6}\text{A}\text{?}$) exists as a bilayer, and the 14 Å series is probably another cholesterol phase.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

The inhibitor effect of probenecid and structural analogues on organic anions and chloride permeabilities in ox erythrocytes

Probenecid inhibits anion movements (organic anions and chloride) in ox erythrocytes. The I₅₀ is 4 · 10^?5 M. Structural analogues such as carinamide, $\text{p-}\text{carboxybenzene}$ sulfonamide and $\text{p-}\text{carboxy}$$\text{N,N}$ diethyl benzene sulfonamide, which are drugs of the sulfonamide class, were also found to inhibit anion transport. These results reinforce the previously discussed view based on structural considerations, that sulfonamides act on the red cell membrane as competitors of anion transport. It is possible that probenecid and carinamide act in a similar way in the kidney.