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PubMed1974

Specific electron donor-energized transport of alpha-aminoisobutyric acid and K+ into intact cells of a marine pseudomonad.

The transport of alpha-aminoisobutyric acid and K(+) into K(+)-depleted cells of a marine pseudomonad (ATCC 19855) was stimulated strongly by ethanol, reduced nicotinamide adenine dinucleotide (NADH), and ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine. In the presence of the quinone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide, only ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine was active. Primary and secondary, but not tertiary, alcohols from ethanol to n-amyl alcohol stimulated both alpha-aminoisobutyric acid and K(+) transport and were oxidized by the cells. Malate and succinate, which were oxidized rapidly by the cells, had little or no capacity to energize the transport of alpha-aminoisobutyric acid into K(+)-depleted cells but were partially effective in promoting K(+) uptake. Ethanol stimulated the transport of alpha-aminoisobutyric acid into K(+)-preloaded cells. The transport of both alpha-aminoisobutyric acid and K(+) was inhibited 20% by iodoacetate, 85% by N-ethylmaleimide, and 90 to 100% by both NaCN and p-chloromercuribenzoate. The addition of Na(3)Fe(CN)(6) permitted the ethanol-induced transport of alpha-aminoisobutyric acid into K(+)-preloaded cells in the presence of NaCN, but little or no uptake of alpha-aminoisobutyric acid or of K(+) into K(+)-depleted cells under the same conditions. The transport of alpha-aminoisobutyric acid into K(+)-depleted cells required both K(+) and an electron donor. The oxidation of NADH and ethanol by K(+)-depleted cells was stimulated strongly by K(+). Parallels between these studies and those with membrane vesicles show that results with membrane vesicles of the marine pseudomonad have physiological significance for the intact cells. The results support the conclusion that the energy for the active transport of both alpha-aminoisobutyric acid and K(+) into cells of this organism is provided by electron flow through a region of the respiratory chain lying between cytochrome c and O(2).

Alcohols↗

PubMed1980

The entry process as the target for energy input in active transport of alpha-aminoisobutyric acid by Mycobacterium phlei.

Mycobacterium phlei was shown to accumulate alpha-aminoisobutyric acid, establishing a concentration gradient of approximately 15,000-fold. The apparent affinity constant of the carrier for alpha-aminoisobutyric acid was 1.8 microM. The system exhibited a broad specificity provided two structural requirements were satisfied: the presence of a free amino and carboxyl group on the alpha carbon and the absence of a net charge. The role of energy coupling on the accumulation of alpha-aminoisobutyric acid was studied by two different kinds of experiments, the relative effects of the inhibitors on the rate of entry and the steady-state of accumulation, and a comparison of the efflux induced at the final steady state by the addition of (a) excess nonradioactive alpha-aminoisobutyric acid, (b) energy inhibitors, or (c) both. The results are consistent with the hypothesis that accumulation of alpha-aminoisobutyric acid is due to an increased rate of entry, the rate of exit not being affected by metabolic inhibitors.

Amino Acids↗

PubMed1978

Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples.

A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.

Alanine↗

PubMed2002

Effect of increased glucose load on maternal-fetal transport of alpha-aminoisobutyric acid in the perfused human placenta: in vitro study.

The role of hyperglycemia on modulation of maternal-fetal transport of amino acids in humans is little understood. Hence, we have explored the effect of increased glucose load on transport kinetics of a model non-metabolizable amino acid, alpha-aminoisobutyric acid (AIB), in the human placenta in vitro. Transport kinetics of AIB in maternal-fetal direction was studied using perfusion of isolated human placental lobules. NCTC (National Culture and Tissue Collection)-135 medium, diluted with Earle's buffered salt solution was used as the perfusate and tritiated water was used as the reference marker. Effect of increased glucose load on transport kinetics of study and reference substances was studied in normal term placentae (n=5; gestational age, 38.5 +/- 0.5 weeks) in succeeding experimental phases, after a control perfusion phase with physiological glucose concentration. AIB transport fraction (TF), relative to tritiated water TF, averaged 54.8% in control euglycemic phase while in hyperglycemic concentration phases of 27.8 and 55.6 mM, the AIB TF index averaged 42.4% and 38.2%, respectively. Analysis of variance revealed that the difference was statistically significant. Similarly, absorption rate index of the amino acid was also significantly lower in the hyperglycemic perfusion phases compared to control euglycemic phase. We conclude that hyperglycemia may play a deleterious role in limiting maternal-fetal transport of A-type amino acids in the in vivo state.

Aminoisobutyric Acids↗

PubMedDate not supplied

Transport kinetics of alpha-aminoisobutyric acid and water in pre-eclamptic pregnancies: an in vitro study.

OBJECTIVE: To investigate the transport kinetics of a model amino acid, alpha-aminoisobutyric acid (AIB), in the maternal-fetal direction in placentae from pre-eclamptic pregnancies. METHODS: Transport kinetics of the amino acid was assessed in vitro using perfusion of human placental lobules. Control placental lobules were perfused for comparison. National Culture and Tissue Collection--135 medium diluted with Earle's buffered salt solution was used as the perfusate, and tritiated water served as the internal reference marker. AIB along with reference marker was injected as a single bolus into the maternal arterial perfusate, and serial perfusate samples were collected from maternal and fetal venous circuits for a study period of 5 min. RESULTS: The differential transport rate of the amino acid for various efflux fractions differed significantly between control and experimental groups when compared to that of the reference marker. The transport rate (corresponding to 50% of efflux into the fetal vein), transport fraction, absorption and elimination rates of the amino acid differed significantly compared to the reference marker values in study and control groups. CONCLUSION: The results indicate that amino acid transport function is compromised in placentae of pre-eclamptic pregnancies.

Adult↗

PubMed1987

beta-Aminoisobutyric acid as a marker of thymine catabolism in malignancy.

Urine from untreated patients with various tumours and controls has been examined for the excretion of beta-aminoisobutyric acid and uric acid. The patients were classified into four groups: I, beta-aminoisobutyric acid and uric acid both normal; II, beta-aminoisobutyric acid normal, uric acid elevated; III, beta-aminoisobutyric acid elevated, uric acid normal; IV, beta-aminoisobutyric acid and uric acid both elevated. Uric acid was used as an indicator for tissue-breakdown. Pseudouridine being a specific parameter for t-RNA degradation was estimated for comparison. Increased urinary concentrations of beta-aminoisobutyric acid were frequently found in tumour patients, especially in patients with leukaemia and non-Hodgkin lymphoma. Tissue breakdown being the cause of the beta-aminoisobutyric aciduria could only be considered in part of the patients. Moreover, strongly elevated ratios of beta-aminoisobutyric acid to uric acid were found. Urinary patterns of pyrimidines and purines were determined in order to exclude other abnormalities. The results are discussed in relation to thymine metabolism and renal function.

Adolescent↗

PubMed1977

The effects of neonatal androgenization on the in vivo transport of alpha-aminoisobutyric acid into specific regions of the rat brain.

The purpose of the present study was to determine if the administration of testosterone propionate (TP) to neonatal rats is followed in vivo by alterations in the transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid (AIB), into specific regions of the brain. Forty-eigh hours after birth, male and female rats were injected s.c. with either 1,25 mg TP or an equivalent volume of vehicle. Five, 10 and 17 days after birth, control and TP-treated rats were decapitated at intervals of 2, 5, 60 and 300 min after the i.p. injection of 0.25 muCi [1-14C]alpha-aminoisobutyric acid/g body weight. Twelve brain regions, i.e., amygdala, cerebellum, corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pons, pyriform cortex and samples of serum were analyzed in terms of disint./min/mg tissue and as tissue/serum (T/S) ratios. At the end of 300 min there was a significant increase in the active transport of AIB in all brain regions of the 5-days-old TP-treated rats. Similarly, by 300 min, the active transport of AIB was significantly increased in all brain regions sxcept cerebellum and pons of the 10-day-old TP-treated rats. The administration of TP to neonatal rats did not alter the accumulation and/or active transport of AIB in brain regions of the 17-day-old rat at any of the tested intervals. These data indicate that (1) neonatally administered TP enhanced (either directly or indirectly) the transport and/or accumuation of AIB in specific brain regions of 5- and 10-day-old rats and (2) the effectiveness of the steroid decreased with the age of the rat.

Aminoisobutyric Acids↗

PubMed2001

Effect of insulin on transport kinetics of alpha-aminoisobutyric acid in the perfused human placental lobule in vitro.

BACKGROUND: The limited data available on the role of insulin on maternal-fetal transport of amino acids prompted us to undertake this study. METHODS: Transport kinetics of a model amino acid, alpha-aminoisobutyric acid (AIB) was investigated in perfusion of isolated human placental lobules in vitro in non steady-state conditions and the effect of therapeutic dose of insulin was assessed in parallel series of perfusions. National Culture and Tissue Collection medium diluted with Earle's buffered salt solution was used as the perfusate and tritiated water was used as the reference marker for comparison. RESULTS: In five successful perfusions with insulin, differential transport rate indices of AIB for 10, 25, 50, 75 and 90% of efflux fractions in the fetal venous outflow averaged 0.76, 1.03, 1.02, 1.09, 1.04 and 1.03 times those of reference values, respectively. The indices differed significantly than in controls for 10, 25 and 50% efflux fractions, but not in the case of 75 and 90% efflux values. The AIB transport fraction (TF), expressed as percentage of injected maternal dose, averaged 29.4 +/- 5.4% and 38.7 +/- 6.2% of the corresponding reference marker value in control and insulin series, respectively. With regards to the pharmacokinetic transport parameters, the absorption and elimination rates of the amino acid were significantly higher in the study group than in the control. CONCLUSION: We conclude that insulin, in physiological and therapeutic doses, stimulates maternal-fetal AIB transport in vitro, in the perfused human placental lobule.

Aminoisobutyric Acids↗

PubMed1991

Acetylcholine output and materno-fetal alpha-aminoisobutyric acid transfer in the perfused human placental lobule.

Dually perfused human placental lobules were employed to study the effects of vesamicol (an inhibitor of acetylcholine (ACh) storage) and physostigmine (an inhibitor of cholinesterase breakdown of ACh) on ACh output from fetal vessels and on materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). Bilateral perfusion with vesamicol (100 mumol L-1) or omission of physostigmine (2 x 7 mumol L-1) from the perfusate significantly reduced the output of ACh. However, neither drug had any significant effect on AIB transfer. Thus, the results of this study do not provide evidence for a close relationship between the placental cholinergic system and materno-fetal AIB transfer.

Acetylcholine↗

PubMed1979

Serum-dependent regulation of alpha-aminoisobutyric acid uptake in bovine granulosa cells.

The removal of serum from the medium of ovarian granulosa cells in exponential or confluent stages of growth results in a rapid and pronounced decrease in the rate of transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid. This decrease is rapidly and completely reversed by the addition of serum. The decrease and its reversal are insensitive to inhibitors of RNA and protein syntheisis and are unaffected by a number of other metabolic inhibitors. The serum requirement cannot be replaced by peptide hormones known to stimulate cell division and secretion by these cells. These data are consistent with a model of post-translational control of AIB transport by a high-molecular-weight component of serum.

Aminoisobutyric Acids↗

PubMed1981

Acetylcholine in human term placenta: tissue levels in intact fragments after inhibition in vitro of choline acetyltransferase and relationship to [14C]alpha-aminoisobutyric acid uptake.

Choline acetyltransferase (ChAc), the enzyme catalysing the biosynthesis of acetylcholine (ACh) in the non-innervated human placenta, was rapidly and persistently inhibited by (2-benzoylethyl)trimethylammonium (BETA) when the drug was applied to intact tissue fragments. This inhibition (50 per cent at congruent to 0.4 mmol/1 BETA) was coupled to a concomitant reduction in the active uptake against a concentration gradient of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). The reduction of AIB accumulation (50 per cent at congruent to 0.1 mmol/1 BETA) was temporally related to inhibition of ChAc. These effects suggest that AIB uptake by the human placenta and ACh biosynthesis catalysed by ChAc are related. Measurements of total ACh content in tissue samples treated in parallel with those destined for ChAc and AIB uptake determinations revealed that BETA (3 mmol/1) significantly reduced the ACh levels by 35 to 50 per cent. This drug concentration caused almost complete inhibition of ChAc and blockade of AIB accumulation.

Acetylcholine↗

PubMed1995

Calcium dependence of alpha-aminoisobutyric acid transfer in human bilaterally perfused placental lobules.

Human isolated bilaterally perfused placental lobules were employed to study the Ca++ dependence of materno-fetal transfer of the non-metabolisable amino acid, alpha-aminoisobutyric acid (AIB). Bilateral perfusion with Ca(++)-free Krebs' solution containing 2 mmol/l EDTA for 60 min resulted in a significant reduction in materno-fetal AIB transfer compared to controls perfused with normal Krebs' solution. However, bilateral perfusion with Krebs' solution containing: 2.5 mmol/l Ca++ and 2 mmol/l EDTA; 3.8 mmol/l Ca++ with no EDTA; or Ca(++)-free Krebs' solution alone did not produce significantly different materno-fetal AIB transfer compared to placentae perfused with normal Krebs' solution. The reduction in AIB transfer during perfusion with Ca(++)-free Krebs' solution with EDTA was partially restored with subsequent reperfusion with normal Krebs' solution for 90 min. These results suggest that AIB transfer in human perfused placental lobules is at least partially Ca(++)-dependent.

Aminoisobutyric Acids↗

PubMed1980

Inhibition of pancreatic alpha-aminoisobutyric acid uptake by cholecystokinin and other secretagogues.

The uptake of the non-metabolizable amino acid, alpha-aminoisobutyric acid (AIB), by isolated mouse pancreatic acini was studied. AIB was concentrated in acinar cell water by an Na+-dependent mechanism. The protein hormone secretagogues caerulein, cholecystokinin, and gastrin and the cholinergic agent carbachol inhibited AIB uptake by greater than 50% of control. The effect of secretagogues on AIB uptake was maximal at hormone concentrations that are slightly higher than those that are maximal for amylase release, but comparable to those concentrations that maximally increase glucose transport by acini. This inhibition of AIB uptake was mediated by both inhibition of AIB influx and stimulation of AIB efflux. In the presence of Ca2+, the Ca2+ ionophore A23187 mimicked the effect of caerulein on AIB uptake. In the absence of Ca2+, control AIB uptake was markedly decreased and both caerulein and A23187 had no further effects. It was concluded, therefore, that secretagogues known to induce enzyme release by an effect on cellular Ca2+ also decrease AIB uptake and that this effect on uptake is most likely mediated by Ca2+.

Aminoisobutyric Acids↗

PubMed1992

Local blood-brain barrier in the newborn rabbit: postnatal changes in alpha-aminoisobutyric acid transfer within medulla, cortex, and selected brain areas.

Postnatal changes in local permeability of the blood-brain barrier to an inert neutral amino acid (alpha-[14C]-aminoisobutyric acid) were investigated in 25 rabbits. The local transfer constant (K) for this tracer was measured with quantitative autoradiographic techniques at postnatal ages of 1, 3, 8, and 17 days, and adult. In adults, the amino acid penetrated the blood-brain barrier poorly in most regions examined (K less than 1 microliter.g-1.min-1) except within and in proximity to structures with a relatively leaky blood-brain barrier such as area postrema and choroid plexus. The rate of tracer entry into "impermeable" regions was seven- to 10-fold greater in 1-day-old rabbits than adults and not dependent on active transport. In young animals, there was a pronounced regional variation in K with the lowest values occurring in white matter and the highest in gray matter such as cerebral cortex, posterior thalamus, and hippocampus. During postnatal development, K decreased (p less than 0.01) with most regions having values near those of adults by 17 days of age. The results indicate that the blood-brain barrier of the newborn rabbit is relatively leaky to a small hydrophilic nonelectrolyte with a distribution that is heterogeneous regionally. Irrespective of age, such blood-borne substances can accumulate in certain brain areas considered to have impermeable vessels (e.g., nucleus tractus solitarii).

Aging↗

PubMed1982

Nerve growth factor-induced stimulation of alpha-aminoisobutyric acid uptake in PC12 cells: relationship to plasma membrane receptor occupancy.

Nerve growth factor (NGF) stimulates the uptake rate of the nonmetabolized amino acid alpha-aminoisobutyric acid (AIB) in the clonal PC12 pheochromocytoma cell line by 40-70%. This effect reaches a maximum after a 1-hour incubation with the hormone and then drops over 50%, reaching a minimum after 4 hours of NGF administration. Longer exposure to the hormone leads to a gradual rise in stimulation, and by 24 hours, the cells regain about 80% of the original 1-hour rate. Results of NGF-binding studies indicate that stimulation of AIB uptake follows closely behind the amount of NGF bound to the low-affinity NGF receptors. Dose-response experiments indicate that full stimulation occurs biphasically. Within the NGF concentration range of 0.1 ng/ml to 1 ng/ml, the AIB uptake rate is 30% of the maximum 1-hour response. At 2.5 ng/ml NGF, the stimulation jumps to about 75% maximal response while 100% response is reached between 5 ng/ml and 50 ng/ml NGF. We suggest that the two states of the NGF plasma membrane receptor on PC12 cells may both be involved in mediating NGF stimulation of AIB uptake.

Absorption↗

PubMed1971

The rapid induction by phytohemagglutinin of increased alpha-aminoisobutyric acid uptake by lymphocytes.

The effect of phytohemagglutinin (PHA) on the ability of human lymphocytes to transport the nonutilizable amino acid, alpha-aminoisobutyric acid (AIB) has been studied. PHA binds rapidly to plasma membrane receptor sites with half maximal binding requiring approximately 7.5 min. During the first 30 min after PHA addition to lymphocytes no change was detected in AIB transport, but then a linear increase in the initial rate of AIB transport occurred over the next 9 hr. Subsequently, the rate of AIB transport stabilized at a level 6-7 times greater than that found in control lymphocytes. The change in membrane function developed even when de novo protein synthesis was inhibited by 85-90% with puromycin or cycloheximide. However, the PHA effect did not occur when the lymphocytes were maintained at 4 degrees C. Studies of the kinetics of AIB uptake by control and PHA-treated lymphocytes demonstrated that PHA increases the V(max) of AIB uptake by 6-7-fold (0.7 mmumole AIB per 10(6) lymphocytes/15 min versus 0.1 mmumole per 10(6) lymphocytes/15 min) without affecting the Km (Michaelis constant) of the transport system (2mM in both cases).When fetuin was added to lymphocyte cultures to remove bound PHA, the PHA-induced increase in the rate of AIB uptake was arrested at the rate achieved during the time of prior incubation with PHA. This level of AIB transport persisted for at least 3 hr after 80% of the PHA was removed from the cell membrane. These data demonstrate that PHA rapidly induces a change in a lymphocyte cell membrane transport function, and that the continued presence of PHA on the cell membrane is required for the full stimulatory effect to be reached. The data do not distinguish between a direct action of PHA upon the lymphocyte membrane or the possibility that PHA slowly enters into the cell where it then exerts its effect.

Alpha-Globulins↗

PubMed1980

Alpha-aminoisobutyric acid transport in rat soleus muscle and its modification by membrane stabilizers and insulin.

The non-metabolizable amino acid alpha-aminoisobutyric acid (AIB) has been used to study the effects of insulin and a number of membrane stabilizers on amino acid transport in rat soleus muscle in an attempt to characterize the mechanisms present. 2. Insulin (5--100 millimicron./ml) increases the net uptake of AIB two- to threefold. Since insulin is without significant effects on AIB efflux, stimulation of net uptake appears to result directly from an increased AIB influx. 3. All classes of membrane stabilizer tested affected AIB fluxes but the responses observed varied for different classes of compounds. 4. The total anaesthetic, tetracaine, reduced AIB accumulation both in the absence and presence of insulin by a similar proportion. The effects on AIB efflux were dependent on the concentration of tetracaine used. Efflux was suppressed by concentrations up to 1 mM whereas 4 mM-tetracaine caused a massive stimulation of AIB efflux. Other local anaesthetics and barbiturates produced similar effects. 5. Another group of membrane stabilizers, exemplified by chlorpromazine, also suppressed AIB uptake, but at no concentration did they reduce AIB efflux. In fact, efflux of AIB began to be increased at concentrations of chlorpromazine which were giving only a modest inhibition of uptake. 6. Measurement of the initial rate of uptake showed that it involved two components. Tetracaine appears to inhibit the saturable component whilst leaving the non-saturable component relatively unaffected. 7. The active uptake of AIB was shown to be Na+-dependent, and under Na+-free conditions tetracaine had no effect on the initial rate of uptake. The non-saturable component was also shown to be Na+-sensitive, uptake from high extracellular concentrations of AIB being reduced in Na+-free media. 8. The possibility of the presence of a carrier-mediated AIB efflux mechanism was investigated. AIB efflux was stimulated by extracellular AIB (homo-exchange) or glycine (hetero-exchange), but not mannitol. This suggests the involvement of a carrier-mediated process in AIB efflux. 9. The present study has demonstrated a heterogeneity among different classes of membrane stabilizers in their actions on AIB efflux which is in marked contrast to previous observations of sugar and cation transport in this preparation. Possible reasons for these differences are discussed.

Aminoisobutyric Acids↗

PubMed2001

Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses via the juxtamembrane region of the PTH-1 receptor.

The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21)NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.

Aminoisobutyric Acids↗