Role of ATP and beta-gamma-iminoadenosinetriphosphate in the stimulation of epinephrine and protein release from isolated adrenal secretory vesicles.

ATP-stimulated release of epinephrine and protein from isolated chromaffin granules of the bovine adrenal medulla has been characterized with respect to pH optimum, substrate requirements, and temperature. Chromaffin granules incubated at 37 °C under optimal conditions released virtually all of their epinephrine and soluble protein within 10 min. ATP-stimulated epinephrine release was optimal at pH 5.8–6.2 and was five times greater than at pH 7.0–7.4. Magnesium and chloride were absolutely required for this process. Magnesium stimulated release at concentrations up to 1.0 mm; however, it was moderately inhibitory at higher concentrations. The dependence of release on Cl^? exhibited positive cooperativity and was nonsaturable. At 90 mm Cl^? and 1.0 mm Mg²⁺, ATP-stimulated epinephrine release followed Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.2 mm. The release of soluble chromaffin granule proteins closely paralleled epinephrine release under all conditions tested, while membrane components were not released. Analogs of ATP substituted at the β-γ position with methylene or imino groups were also capable of stimulating release from granules. These ATP analogs had reduced affinity and lower activity than ATP itself. ATP analogs substituted at the α-β position were essentially inactive but were potent inhibitors of ATP-stimulated release. We conclude that the regulation of release from granules by ATP is rapid and specific and may not depend on hydrolysis of ATP at the β-γ position. This ATP-dependent reaction may be involved in the cellular process of exocytosis.     

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.     

Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. β-ν-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.     

The influence of beta-LPH fragments on alpha-MSH release: the involvement of a dopaminergic system

β-Endorphin was able to enhance plasma α-MSH levels in rats after intracerebroventricular injection. This effect could be inhibited by naloxone or by removing tyrosine from position 61 of the peptide. Neither α- and γ-endorphin nor their des-tyrosine analogs appeared to be able to modify plasma α-MSH levels. The stimulating effect of β-endorphin on plasma α-MSH levels could be completely blocked by a simultaneous injection of apomorphine, in an amount in which apomorphine itself had no effect on α-MSH levels in plasma. A single injection of haloperidol increased plasma α-MSH levels in a dose related manner. A dose of haloperidol, which caused an apomorphine antagonizable increase in plasma α-MSH, did not modify β-endorphin elevated α-MSH levels. A high concentration of haloperidol was able to stimulate the basal release of α-MSH from isolated pituitaries $\text{in bitro}$, whereas β-endorphin appeared to be inactive in this respect.These observations indicate a central opiate receptor-mediated influence of β-endorphin on α-MSH release and the possible involvement of a dopaminergic system, mediating the β-endorphin effect.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.     

The isolation and characterization of the methyl acceptor protein from adrenal chromaffin granules

A methyl acceptor protein (MAP), which serves as a substrate for adrenal medullary protein carboxymethylase (PCM, E.C. 2.1.1.24), has been isolated from a hypotonic lysate of adrenal chromaffin granules. The isolated MAP was shown to be distinct from the adrenal chromaffin granule protein, dopamine β-hydroxylase (DBH). The properties of MAP, including its amino acid composition, were comparable to those reported for chromogranin A, a major acidic protein found in adrenal chromaffin granules.     

Structural changes of human RNase L upon homodimerization investigated by Raman spectroscopy

RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-β structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.     

Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Selective suppression of blastogenesis induced by different mitogens: effect of noncyclic adenosine-containing compounds

Lymphocyte responses to the galactosyl binding lectins, SBA and PNA, and responses to Con A are suppressed by cAMP analogs and agents that increase intracellular cAMP to a greater extent than are responses to PHA. Adenosine shares with cAMP this selective inhibitory effect. ATP inhibits lymphocyte proliferation via generation of adenosine. This conclusion is based on the findings that (i) β,γ-methylene ATP (β,γ-met ATP) is similar to ATP in its inhibitory effects, whereas α,β-met ATP has little effect on lymphocyte proliferation, (ii) α,β-met ATP reverses both ATP and β,γ-met ATP induced suppression, but does not reverse adenosine-mediated suppression, and (iii) inhibition of adenosine deaminase potentiates ATP and β,γ met ATP-mediated suppression of blastogenesis. The relative potency of various noncyclic adenosine compounds in suppressing mitogen-induced blastogenesis is: 2-chloradenosine > ATP ≥ ADP > AMP > adenosine.     

Active proton uptake by chromaffin granules: observation by amine distribution and phosphorus-31 nuclear magnetic resonance techniques.

The hydrogen ion activity within isolated chromaffin granules can be estimated from the distribution of the weak base methylamine and from phosphorus-31 nuclear magnetic resonance spectra of ATP contained in the granules. Following the addition of ATP to the external medium, the internal pH drops by 0.2 to 0.5 unit. This change occurs only in medium containing a permeant anion such as chloride and is abolished by an uncoupler of oxidative phosphorylation. These results indicate that the chromaffin granule membrane possess an electrogenic proton pump directed inward.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

DNA sequence selectivity of hairpin polyamide turn units

A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a β-amine residue at the turn unit, showed improved binding affinities relative to their α-amino-γ-turn analogs for particular sequences. We incorporated β-amino-γ-turns in six-ring polyamides and determined whether there are any sequence preferences under the turn unit by quantitative footprinting titrations. Although there was an energetic penalty for G·C and C·G base pairs, we found little preference for T·A over A·T at the β-amino-γ-turn position. Fluorine and hydroxyl substituted α-amino-γ-turns were synthesized for comparison. Their binding affinities and specificities in the context of six-ring polyamides demonstrated overall diminished affinity and no additional specificity at the turn position. We anticipate that this study will be a baseline for further investigation of the turn subunit as a recognition element for the DNA minor groove.