Inhibition of anion transport in human red blood cells by 5,5′-dithiobis(2-nitrobenzoic acid)

The uptake of [³²P]phosphate into human red blood cells was inhibited ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.6}\text{mM}$) by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7}\text{mM}$). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[¹⁴C]-benzamido-4′-aminostilbene-2,2′-disulfonate, a potent inhibitor of anion transport ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1?2 μ}\text{M}$), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.     

Kinetic mechanism of chlorpromazine inhibition of erythrocyte 3-O-methylglucose transport

The kinetic mechanism of chlorpromazine inhibition of erythrocyte hexose transport was investigated using the non-metabolizable glucose analog $\text{3-O-}\text{methylglucose}$. It was found that chlorpromazine added to the external medium is a non-competitive inhibitor of both equilibrium exchange and net $\text{3-O-}\text{methylglucose}$ transport at pH 7.8, 15°C. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for equilibrium exchange is $\text{76 ± 21 μM}$. When net efflux and equilibrium exchange were measured on the same population of cells the equilibrium exchange was 2.5-times the maximum net efflux. The percent reduction of $\text{3-O-}\text{methylglucose}$ flux by chlorpromazine is dependent upon chlorpromazine concentration and not $\text{3-O-}\text{methylglucose}$ concentration as expected for a non-competitive inhibitor. Equilibrium exchange and net efflux show the same extent of inhibition at each concentration of chlorpromazine evaluated. These results suggest that exchange and net efflux of $\text{3-O-}\text{methylglucose}$ in the human erythrocyte may share a common transport system.     

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit ⁵⁹Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis $\text{in}$$\text{vitro}$ and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

An inhibitor of protein synthesis prepared by protease treatment of mouse cerebral cortex cells

A substance was isolated from mouse brain cortical tissue that inhibits both cell division and protein synthesis by cells in culture. The inhibitor was released from cerebral cortex tissue by mild protease treatment. A single exposure of cells to as little as 1.25 μg of the isolated material was sufficient to inhibit BHK-21 cell protein synthesis by 20%. Higher concentrations and continual exposure resulted in 87% reduction in protein synthesis. The inhibition was shown to be independent of amino acid uptake and most effective against primary mouse embryo fibroblasts and neonatal mouse brain cell suspensions. Cells previously adapted to culture or transformed cells derived from the nervous system were less affected by, or refractory to, the inhibitor. The substance was shown to be nondialyzable, relatively resistant to thermal inactivation and the inhibitor activity was not removed by chloroform extraction. Two active fractions were identified by Bio-Gel P-100 chromatography and the protein synthetic inhibitor was removed by affinity chromatography with $\text{Ulex}$$\text{europus}$ agglutinin.     

Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpiusrotundacauda

The sialic acid binding loctin carcinoscorpin agglutinates $\text{Escharichia}\text{coli}\text{K12}\text{and}\text{Salmonella}\text{minnesots}$ R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. $\text{Vibrio}\text{cholerae}$, a Grum negative bectarium devoid of 2-keto-3-deoxyoctonate and $\text{Staphylococcus}\text{sureus}$ a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoa crab.     

Photoinactivation of the thiamine transport system in saccharomyces cerevisiae with 4-azido-2-nitrobenzoylthiamine

A newly synthesized photoreactive thiamine derivative, 4-azido-2-nitrobenzoylthiamine was found to be a competitive inhibitor of the thiamine transport system in Saccharomyces cerevisiae, exhibiting an apparent $\text{K}{}_{\mathrm{i}}$ of 36 nM. When exposed to visible light, 4-azido-2-nitrobenzoylthiamine irreversibly inactivated the thiamine transport. 4-azido-2-nitrobenzoylthiamine-dependent photoinactivation of thiamine transport was partially protected by thiamine, but not by the nitrene-trapping reagent $\text{p-}\text{aminobenzoate}$. On the other hand, the irradiation of the yeast cells in the presence of 4-azido-2-nitrobenzoylthiamine did not significantly lead to inactivation of the biotin transport system. The results suggest that 4-azido-2-nitrobenzoylthiamine is a specific irreversible inhibitor of the thiamine transport system in Saccharomyces cerevisiae.     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

Discordant regulation by luteinizing hormone of ornithine decarboxylase activity and testosterone production in isolated rat testicular cells invitro

Possible functional relationship between luteinizing hormone-stimulated ornithine decarboxylase and testosterone production was examined in rat testicular interstitial cells $\text{in}\text{vitro}$. Although luteinizing hormone enhanced both ornithine decarboxylase activity and testosterone production at a similar physiological dose range, we found dissociation in the two responses in terms of their temporal aspect and the way they were affected by an irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, and protein synthesis inhibitor cycloheximide. The results suggest that there appears to be no causal coupling between luteinizing hormone-stimulated enzyme activity and testicular steroidogenesis.     

Effects of netropsin on yeast mitochondria

Netropsin binds tightly to AT rich regions of DNA and correspondingly is an efficient inhibitor of mitochondrial DNA replication in $\text{Saccharomyces}\text{cerevisiae}$. Netropsin treatment does not cause formation of large populations of petite cells. However, a large portion of cells grown in cultures with ethanol as carbon source are killed by 1 μg/ml netropsin. When petite induction by berenil or ethidium bromide is carried out in the presence of netropsin, the petite cells are killed. This appears to be an effect of netropsin action on the cells during the process of petite formation.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

The effect of tosyl lysine chloromethyl ketone on the activity of uridine diphosphoglucose pyrophosphorylase of the cellular slime mold Dictyostelium discoideum

We report here that the specific activity of UDPG pyrophosphorylase in extracts of $\text{D. discoideum}$ amoebae and preculmination-stage cells increases as a function of the length of their exposure to tosyl lysine chloromethyl ketone, an irreversible inhibitor of a number of serine and sulfhydryl proteases. This compound also stabilizes the activity of the enzyme in crude extracts of amoebae. These results can be interpreted, with some assumptions, as evidence in support of the hypothesis that the levels of the enzyme are maintained in $\text{D. discoideum}$ by a balance of synthesis and degradation.     

A trypsin and chymotrypsin inhibitor from Taenia pisiformis

The somatic extract of mature T. pisiformis has been demonstrated to contain a potent inhibitor capable of inactivating the esterolysis of $\text{N-α-}\text{benzoyl-}\text{L}\text{-arginine ethyl ester}$ and $\text{N-}\text{benzoyl-}\text{L}\text{-tyrosine ethyl ester}$ by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the caseinolytic activity of subtilisin and elastase. The protease inhibitor, partially purified by trichloroacetic acid treatment, Sephadex G-100 column chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine chymotrypsin conjugate, was soluble in 5% trichloroacetic acid, stable to heating at 100°C for up to 30 min, tolerated the pH range of 1.5–9.0, and was unaffected by 8 m-urea or 0.2 M-2-mercaptoethanol. The molecular weight of the inhibitor was estimated to be 7000–7200 by Sephadex G-100 chromatography. Activity determinations on crystalline bovine trypsin and chymotrypsin revealed that both inhibitory actions are located on the same or closely adjacent sites of the inhibitor molecule. Complex formation between the inhibitor and mammalian trypsin and chymotrypsin required 3–4 min for completion.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Occurrence of proteinase a isoinhibitors in wild type yeast strains and commercial baker's yeast

The occurrence of the proteinase A inhibitors 2 and 3 was investigated in wild type strains of $\text{Saccharomyces}\text{cerevisiae}$ and $\text{Saccharomyces}\text{carlsbergensis}$ as well as in several strains of commercial baker's yeast. Haploid and diploid strains of $\text{Saccharomyces}\text{cerevisiae}$ contain only proteinase A inhibitor 3 whereas in $\text{Saccharomyces}\text{carlsbergensis}$ only proteinase A inhibitor 2 is found. Strains of commercial baker's yeast contain either proteinase A inhibitor 3 or both inhibitors in a constant ratio of 1:3. Single cell cultures isolated from a strain of commercial baker's yeast also contain a mixture of the two inhibitors. Therefore, baker's yeast is not a mixture of two different cell types but the genome for both inhibitors is present in each single cell. In general, the results indicate that the occurrence of the two proteinase A inhibitors is determined genetically and, therefore, they may be called “isoinhibitors”.