Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Anomalous enhancing effect of hydroxyurea on DNA synthesis

$\text{In vitro}$ cultures of $\text{Crithidia}$ sp. were exposed to various concentrations of hydroxyurea (HU) during the logarithmic phase. In the presence of 5 × 10^?2M HU, cell division was completely blocked after an initial increase in cell numbers by about 20%. Inhibition of incorporation of ³H-thymidine into acid-insoluble material was effective within 1 hr of exposure to the drug (5 × 10^?2M) and it reached a level of 80% after 8 hr. At lower concentrations (5 × 10^?4M ? 1 × 10^?3M), however, incorporation of ³H-thymidine was remarkably increased while cell division remained unaffected indicating that the increase in incorporation was not due to increased DNA synthesis in preparation for cell division.     

Studies on two isozymes of aconitase from Bacillus cereus T. III. Enzymatic properties.

The present communication describes a comparative study of some enzymatic properties of an early and a late aconitase (EC.4.2.1.3) present in $\text{Bacillus}\text{cereus}$ T cells of 5 and 12 hr culture age, respectively. The activity of both enzymes increased linearly with increase in enzyme concentration. They demonstrated similar pH *7.5) and temperature (30 C) optima, but differed in their activation energy and affinity for substrate. Late aconitase had higher activation energy (16,100 cal) as compared to early aconitase (9,200 cal). Early aconitase showed a Km value of 100 × 10^?4M for sodium citrate and 33.3 × 10^?4M for isocitrate. Late aconitase exhibited 5 to 7 times greater affinity for citrate and isocitrate yielding Km values 14 × 10^?4M and 7 × 10^?4M, respectively. On the basis of available evidence, it is suggested that early and late aconitase present in 5 and 12 hr aged cells of $\text{Bacillus}\text{cereus}$ T behave as isozymes, and may be designated as aconitase (EC.4.2.1.3) isozyme I and aconitase (EC.4.2.1.3) isozyme II, respectively. The significance of their plausible role during growth and sporulation has been discussed.     

Inhibition of spider mite ATPases by plictran and three organochlorine acaricides

The ATPase enzyme system from two-spotted spider mites, $\text{Tetranychus}$$\text{urticae}$ (Koch) was sensitive, in vitro, to four acaricides. Tricyclohexylhydroxytin (Plictran^R) was an outstanding inhibitor of oligomycin-sensitive (mitochondrial) Mg²⁺ATPase from fish brain and spider mite homogenates. The I₅₀ values were 6.6×10^?11M and 6.2×10^?10M, respectively. Less effective were chlorbenside, chlorfenethol and ovotran. Plictran at a higher concentration (2×10^?7M) was also more effective on Na⁺-K⁺ATPase both in mites and fish brain homogenates as compared to chlorfenethol, chlorbenside and ovotran. Plictran inhibited oligomycin-insensitive Mg²⁺ATPase at concentrations of 10^?8M but stimulated at high concentrations (5×10^?6M and higher).     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

A new antibiotic with known resistance factors,G418, inhibits plant cells

Growth rates of two lines of tobacco ($\text{Nicotiana}\text{tabacum}$) cell suspension cultures were measured in the presence or absence of G418, a new 2-deoxystreptamine antibiotic related to Gentamycin. Cell growth rates of $\text{N}$. $\text{tabacum}$ cv. Burley were inhibited at drug concentrations as low as 1.65 × 10^?7 M. At 4 × 10^?7 M, the doubling time was increased from 1.5 days (control) to 2.3 days (treatment). The drug was lethal to cells at 4 × 10^?6 M, and inhibition was irreversible. Cells of $\text{N}$. $\text{tabacum}$ cv. Wisconsin 38 also were inhibited by the drug, although at slightly higher concentrations (ca. 2–5 fold).In view of our findings, G418 and its associated resistance factors could be of great value in plant genetic engineering.     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Plasmodium lophurae: Quantitative in vitro incorporation of 14C-1-acetate into lipids

Blood from ducks parasitized with Plasmodium lophurae and normal duck blood were incubated with sodium ¹⁴C-1-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and plasma and lipid classes separated by thin-layer chromatography. Specific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated ¹⁴C-labeled lipid precursors.Experiments employing sodium ¹⁴C-1-acetate in two concentrations, 50 μCi ¹⁴C in 0.91 μmole sodium acetate/50 ml blood and 500 μCi ¹⁴C in 9.1 μmole sodium acetate/50 ml blood (1.82 × 10^?5M and 1.82 × 10^?4M), showed higher ¹⁴C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At $\text{1.82 × 10}{}^{\mathrm{?5}}\text{M}{}^{14}$C-1-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration (1.82 × 10^?4M), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. lophurae.Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols.At 24 hr of incubation, the lipids of partially purified P. lophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides.In plasma from parasitized blood incubated 24 hr with ¹⁴C-1-acetate, the highest specific activity and greatest percent of ¹⁴C incorporation was found in free fatty acids.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10^?2 min^?1. This provides a $\text{t}\text{1}\text{2}$ of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10^?2 min^?1 and 7 × 10^?3 min^?1 for uncatalyzed autoxidation and 4 × 10^?2 min^?1 and 1.1 × 10^?2 min^?1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

The preferential human mononuclear leukocyte chemotactic activity of the substituent tetrapeptides of angiotensin II

The amino- and carboxy-terminal substituent tetrapeptides of angiotensin II, Asp-Arg-Val-Tyr and Ile-His-Pro-Phe, elicit substantial human mononuclear leukocyte chemotactic responses $\text{in}\text{vitro}$ that attain maximal levels at tetrapeptide concentrations of 3 × 10^?8 M and 3 × 10^?7 M, respectively. In contrast, the angiotensin II-derived tetrapeptides evoke only marginal human neutrophil chemotactic responses. Amino acid deletions or substitutions that alter the properties of the tetrapeptides, reduce their chemotactic potency and activity. Limited proteolytic cleavage of angiotensin II thus may convert a pathway with predominantly humoral effects to a source of mediators that regulate cellular immunity and chronic inflammatory responses.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Stimulative effect of guanylylimidodiphosphate on the vasopressin-induced increment of osmotic water flow of the toad bladder.

The effect of guanylylimidodiphosphate [Gpp(NH)p] on vasopressin-induced osmotic water flow across the bladder of the toad, $\text{Bufo}\text{bufo}\text{japonicus}$ was examined. Gpp(NH)p significantly enhanced vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M, while it showed no effect on the water flow without vasopressin. Gpp(NH)p alone could not enhance cyclic AMP-induced osmotic water flow of the toad bladder. Adenylylimidodiphosphate [App(NH)p] could not enhance vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M. The results suggest that Gpp(NH)p can enhance the physiological effect of vasopressin by stimulating vasopressin activation of adenylate cyclase during substrate and hormone depletion of the toad bladder.     

Bioreduction of nitroxides by Staphylococcus aureus.

Two nitroxide radicals (TEMPO, I; OXAN, II) and a spin labeled penicillin (III) were reduced by $\text{Staphylococcus aureus}$. A short induction period preceded zero order reduction of these substrates leading to a K_m of 8 × 10^?4M, 6.67 × 10^?5M and 5.7 × 10^?4M and V_max of 106, 26 and 11 μ mole/min mg bacteria for I, II and III, respectively.     

Evidence for selective sulfhydryl reactivity in cytochalasin A--mediated bacterial inhibitions.

Cytochalasin A (CA) at 5 × 10^?5$\text{M}$ strongly inhibits glucose transport in Arthrobacter sialophilis. This effect and other bacteriostatic and metabolic inhibitions of gram-positive bacteria are not caused by the closely related congeners cytochalasin B or D. Inhibitions by CA are nullified by prior drug incubation with sulfhydryl compounds. It was also found that the characterized adduct of CA with β-mercaptoethanol is devoid of biological activity. N-ethylmaleimide, p-chloromercuribenzoate and ethacrynic acid (a known, liposoluble, sulfhydryl reactant) were each shown at 5 × 10^?5$\text{M}$ to be relatively ineffective in inhibiting D-glucose transport in $\text{A. sialophilus}$. These observations suggest that CA reacts at the molecular biological level in a site-specific manner.