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Biomedical subjects

A P Hansen

Publications and source records attributed to A P Hansen.

At least 19 recordsLinked to original sources
PubMed2005

Inactivation of penicillin G in milk using hydrogen peroxide.

Milk antibiotic residues have been a public concern in recent years. The Grade A Pasteurized Milk Ordinance mandates that raw Grade A milk will test negative for beta-lactam antibiotic residues before processing. The purpose of this research was to investigate the ability of various levels of peroxide and heat to inactivate penicillin G in raw milk. Whole milk spiked to a mean of 436 +/- 15.1 (standard error of the mean) ppb of potassium penicillin G was treated with hydrogen peroxide at levels of 0.0, 0.09, 0.17, and 0.34%. Samples at each peroxide level (n = 6 per treatment) were treated as follows: 1) incubated at 54.4 degrees C for 3 h, 2) pasteurized at 62.8 degrees C for 30 min, 3) incubated and pasteurized as in treatments 1 and 2, or 4) received no further treatment. A beta-lactam competitive microbial receptor assay was used for quantification of penicillin G. Concentrations of penicillin in selected samples were determined by HPLC for a comparison of test methods. Treatments were evaluated relative to their ability to reduce milk penicillin G levels to below the safe level of 5 ppb. The 0.09% hydrogen peroxide level was ineffective for all treatments. Hydrogen peroxide at 0.17% lowered the mean penicillin G (+/- SEM) from 436 +/- 15.1 to 6 +/- 1.49 ppb using the incubated and pasteurized heat treatment. The 0.34% concentration of hydrogen peroxide was the most effective, inactivating penicillin G to a level well below the safe level of 5 ppb with the pasteurized heat treatment, with or without incubation.

Animals↗
PubMed2001

Acute and short-term administration of a sulfonylurea (gliclazide) increases pulsatile insulin secretion in type 2 diabetes.

The high-frequency oscillatory pattern of insulin release is disturbed in type 2 diabetes. Although sulfonylurea drugs are widely used for the treatment of this disease, their effect on insulin release patterns is not well established. The aim of the present study was to assess the impact of acute treatment and 5 weeks of sulfonylurea (gliclazide) treatment on insulin secretory dynamics in type 2 diabetic patients. To this end, 10 patients with type 2 diabetes (age 53 +/- 2 years, BMI 27.5 +/- 1.1 kg/m(2), fasting plasma glucose 9.8 +/- 0.8 mmol/l, HbA(1c) 7.5 +/- 0.3%) were studied in a double-blind placebo-controlled prospective crossover design. Patients received 40-80 mg gliclazide/placebo twice daily for 5 weeks with a 6-week washout period intervening. Insulin pulsatility was assessed by 1-min interval blood sampling for 75 min 1) under baseline conditions (baseline), 2) 3 h after the first dose (80 mg) of gliclazide (acute) with the plasma glucose concentration clamped at the baseline value, 3) after 5 weeks of treatment (5 weeks), and 4) after 5 weeks of treatment with the plasma glucose concentration clamped during the sampling at the value of the baseline assessment (5 weeks-elevated). Serum insulin concentration time series were analyzed by deconvolution, approximate entropy (ApEn), and spectral and autocorrelation methods to quantitate pulsatility and regularity. The P values given are gliclazide versus placebo; results are means +/- SE. Fasting plasma glucose was reduced after gliclazide treatment (baseline vs. 5 weeks: gliclazide, 10.0 +/- 0.9 vs. 7.8 +/- 0.6 mmol/l; placebo, 10.0 +/- 0.8 vs. 11.0 +/- 0.9 mmol/l, P = 0.001). Insulin secretory burst mass was increased (baseline vs. acute: gliclazide, 43.0 +/- 12.0 vs. 61.0 +/- 17.0 pmol. l(-1). pulse(-1); placebo, 36.1 +/- 8.4 vs. 30.3 +/- 7.4 pmol. l(-1). pulse(-1), P = 0.047; 5 weeks-elevated: gliclazide vs. placebo, 49.7 +/- 13.3 vs. 37.1 +/- 9.5 pmol. l(-1). pulse(-1), P < 0.05) with a similar rise in burst amplitude. Basal (i.e., nonoscillatory) insulin secretion also increased (baseline vs. acute: gliclazide, 8.5 +/- 2.2 vs. 16.7 +/- 4.3 pmol. l(-1). pulse(-1); placebo, 5.9 +/- 0.9 vs. 7.2 +/- 0.9 pmol. l(-1). pulse(-1), P = 0.03; 5 weeks-elevated: gliclazide vs. placebo, 12.2 +/- 2.5 vs. 9.4 +/- 2.1 pmol. l(-1). pulse(-1), P = 0.016). The frequency and regularity of insulin pulses were not modified significantly by the antidiabetic therapy. There was, however, a correlation between individual values for the acute improvement of regularity, as measured by ApEn, and the decrease in fasting plasma glucose during short-term (5-week) gliclazide treatment (r = 0.74, P = 0.014, and r = 0.77, P = 0.009, for fine and coarse ApEn, respectively). In conclusion, the sulfonylurea agent gliclazide augments insulin secretion by concurrently increasing pulse mass and basal insulin secretion without changing secretory burst frequency or regularity. The data suggest a possible relationship between the improvement in short-term glycemic control and the acute improvement of regularity of the in vivo insulin release process.

Blood Glucose↗
PubMed2001

Effect of various dairy packaging materials on the shelf life and flavor of pasteurized milk.

Milk from three different dairies (each a separate trial: 1, 2, and 3) was standardized to 2% fat and pasteurized at 92.2, 84.0, and 76.4 degrees C (temperatures 1, 2, and 3, respectively) for 25 s and packaged into six different packaging boards, [standard (A) milk boards with standard seam; juice boards with standard (B) and J-bottom (D) seams; barrier boards with standard (C) and J-bottom (E) seams; and foil (F) boards with J-bottom seam], resulting in 18 different treatments. Standard plate count (SPC) was used to test for microbial quality, and taste a panel was employed for flavor acceptability and difference on the milk stored at 6.7 degrees C at 1, 2, 3, and 4 wk. Statistical analysis of taste panel data showed that the flavor of milk samples A2, B2, and D2 deteriorated faster than the blind control (freshly high temperature, short time pasteurized low fat milk processed at 80.6 degrees C for 25 s). The flavor of milk packaged in standard (A) and juice (B and D) boards deteriorated at a faster rate than milk packaged in barrier (C and E) and foil (F) boards. Microbial counts showed that milk samples stored at 6.7 degrees C in trials 2 and 3 produced high SPC at wk 3 (ranges of bacteria in cfu/ml for trial 2: 9.9 x 10(1)-1.8 x 10(6) and trial 3: 2.5 x 10(5)-5.5 x 10(8)). In trial 1, high SPC began at wk 4 (9.9 x 10(1)-5.5 x 10(5) cfu/ml). Milk processed at 76.4 degrees C had the lowest bacterial growth rate, and milk processed at 84.0 degrees C had the highest bacterial growth rate. Different boards had no effects (P > 0.05) on the bacterial growth rates. It appeared that the lower the SPC of the raw milk, the slower the bacterial growth rate after 2 wk of storage. Milk samples stored at 1.7 degrees C maintained low SPC at wk 4, with counts of 0 to 40 cfu/ml for trial 2 and 0 to 200 cfu/ml for trial 3.

Animals↗
PubMed2001

Effect of various dairy packaging materials on the headspace analysis of ultrapasteurized milk.

Milk from three different dairies (each a separate trial: 1, 2, and 3) was standardized to 2% fat and processed at 140.6, 129.4, 118.3, and 107.2 degrees C (temperatures 1, 2, 3, and 4, respectively) for 2 s and packaged into six different packaging boards [standard (A) milk boards with standard seam, juice boards with standard (B) and J- bottom (D) seams, barrier boards with standard (C) and J-bottom (E) seams, and foil (F) boards with J-bottom seam] resulting in 24 different treatments. A Shimadzu 15A series chromatograph equipped with a Porapak-P column was used to measure the headspace of the milk stored at 6.7 degrees C for 1, 2, 3, 5, 10, and 15 wk of storage. Gas chromatographic headspace analysis for sulfur compounds showed that hydrogen sulfide, methanethiol, and dimethyl sulfide were detected in milk processed at 140.6, 129.4, 118.3, and 107.2 degrees C. In addition, dimethyl disulfide was detected in milk processed at 140.6 and 129.4 degrees C, and dimethyl trisulfide was detected at 140.6 degrees C. Milk processed at 140.6 degrees C contained the most sulfur compounds. Samples C1, E1, and F1 retained the most hydrogen sulfide and methanethiol at 6 d of storage. Methanethiol appeared to be heat-induced. At wk 6, a slightly hammy or cardboardy flavor was detected for milk packaged in boards with standard seams (A, B, and C), and a slightly cooked flavor was detected for milk packaged in barrier and foil boards with J-bottom (E and F) seams. The hammy or cardboardy flavor intensified with storage time, and all of the cooked flavor dissipated at wk 10.

Animals↗
PubMed2001

Effect of various dairy packaging materials on the shelf life and flavor of ultrapasteurized milk.

Raw milk from three different dairies (each a separate trial: 1, 2, and 3) was standardized to 2% fat and processed at 140.6, 129.4, 118.3, and 107.2 degrees C (temperatures 1, 2, 3, and 4, respectively) for 2 s and packaged into six different packaging boards, [standard (A) milk boards with standard seam, juice boards with standard (B) and J-bottom (D) seams, barrier boards with standard (C) and J-bottom (E) seams and foil (F) boards with J-bottom seam], resulting in 24 different treatments. Standard plate count (SPC) was used to test for microbial quality, and taste panels were employed for flavor acceptability and difference in the milk stored at 6.7 degrees C at 1, 2, 3, 5, 10, and 15 wk. Lipolysis was measured by standard procedures for acid degree value (ADV) of milk. Statistical analysis of taste panel data showed that the flavor of 14 milk samples deteriorated over time. The flavor of UP milk packaged in standard (A) and juice (B and D) boards deteriorated at a faster rate than UP milk packaged in barrier (C and E) and foil (F) boards. At wk 6, a slightly hammy or cardboardy flavor was detected for milk packaged in boards with standard seams (A, B, and C) and a slightly cooked flavor was detected for milk packaged in barrier and foil boards with J-bottom (E and F) seams. The hammy or cardboardy flavor intensified with storage time, and all of the cooked flavor dissipated at wk 10. Milk processed at 118.3 and 129.4 degrees C maintained the lowest bacterial growth rates, and milk processed at 107.2 degrees C had the highest bacterial growth rates during 15 storage wk. More than 87% of milk processed at 118.3, 129.4, and 140.6 degrees C maintained acceptable level of bacterial counts at wk 15. The extent of lipolysis showed that ADV of milk increased with storage time. The ranges of ADV for trials 1, 2, and 3 were 0.76 to 0.85 (from 12 to 22 wk), 0.39 to 0.51 (from 6 to 16 wk), and 0.53 to 0.60 (from 6 to 16 wk), respectively.

Animals↗
PubMed2000

High-frequency oscillations in circulating amylin concentrations in healthy humans.

Amylin is stored in the pancreatic beta-cell granules and cosecreted with insulin in response to nutrient stimuli. To gain further insight into control of hormonal release in beta-cell physiology, we examined whether amylin, like insulin, circulates in a high-frequency oscillatory pattern, and if it does, to compare the secretory patterns of the two hormones. Eight overnight-fasted healthy individuals were studied during intravenous glucose infusion (2.0 mg. kg(-1). min(-1)). Blood was collected every minute for 90 min and analyzed in triplicate for amylin, total amylin immunoreactivity (TAI), and insulin. Mean plasma concentrations of amylin (nonglycosylated), TAI (nonglycosylated plus glycosylated), insulin, and glucose were 2.77 +/- 1.21 pmol/l, 7.60 +/- 1.73 pmol/l, 50.4 +/- 17.5 pmol/l, and 5.9 +/- 0.3 mmol/l, respectively. The 90-min time series of amylin, TAI, and insulin were analyzed for periodicity (by spectral analysis, autocorrelation analysis, and deconvolution analysis) and regularity [by approximate entropy (ApEn)]. Significant spectral density peaks were demonstrated by a random shuffling technique in 7 (out of 7), 8 (out of 8), and 8 (out of 8) time series, respectively, whereas autocorrelation analysis revealed significant pulsatility in 5 (out of 7), 7 (out of 8), and 5 (out of 8), respectively. The dominant periodicity of oscillations determined by spectral analysis was 4.6 +/- 0.3, 4.6 +/- 0.4, and 6. 5 +/- 1.1 min/pulse, respectively (amylin vs. insulin, P = 0.017, TAI vs. insulin, P = 0.018). By deconvolution analysis, amylin and insulin periodicities were, respectively, 6.3 +/- 1.0 and 5.5 +/- 0. 6 min. By application of the regularity statistic, ApEn, 6 (out of 7), 7 (out of 8), and 6 (out of 8), respectively, were found to be significantly different from random. In conclusion, like several other hormones, circulating amylin concentrations exhibit oscillations in the secretory patterns for nonglycosylated as well as glycosylated forms. Whether the high-frequency pulsatile release of amylin is disturbed in diabetes is not known.

Adult↗
PubMed1999

Isolation and characterization of whey phospholipids.

A freeze-dried whey powder was produced by microfiltration of Cheddar cheese whey. A 0.2-micron ceramic membrane in a stainless steel housing unit was used to concentrate components > 400 kDa present in the whey. The experimental whey powder, derived from Cheddar cheese whey, and a commercial whey powder were subjected to proximate analysis, lipid classes, phospholipid classes, and fatty acid compositional analyses. Commercial whey powder and commercial soybean lecithin were subjected to an alcohol fractionation procedure in an effort to alter the ratio of phosphatidyl choline to phosphatidyl ethanolamine and the functionality of dairy phospholipids. The fractionation procedure produced an alcohol-insoluble fraction containing 84% phosphatidyl ethanolamine, whereas the alcohol-soluble fraction resulted in a decrease in the phosphatidyl choline to phosphatidyl ethanolamine ratio. The commercial whey contained a higher ratio of phospholipids to neutral lipids compared with the experimental whey. The classes of phospholipids present within the two wheys were similar, whereas the experimental whey contained a phosphatidyl choline content twice that of the commercial whey, and the phospholipids composition of both wheys differed from the milk fat globule membrane. Comparison of the phospholipids and fatty acid composition of the wheys with the soy lecithin revealed that although the wheys were similar to each other, they differed from the soy lecithin in both the classes of phospholipids present and in the fatty acid composition. These compositional differences may influence the functionality of whey phospholipids.

Calcium↗
PubMed1997

A review of the interactions between milk proteins and dairy flavor compounds.

The effect of sodium caseinate and whey protein concentrate on vanillin, benzaldehyde, citral, and d-limonene was determined by quantitative descriptive analysis deviation from reference. A trained taste panel evaluated samples containing a single flavor compound in 2.5% sucrose solution against a reference sample. Vanillin, benzaldehyde, and d-limonene flavor intensity decreased as the concentration of whey protein concentrate increased. In a separate study, the ability of delipidated methyl ketones to bind straight and branched chain methyl ketones was determined. The concentration of straight chain methyl ketones bound by the milk protein powder was inversely proportional to the size of the ligand. Branched chain methyl ketones did not exhibit a trend in binding based on ligand size.

Animals↗
PubMed1996

Procedures used by North Carolina dairies for vitamins A and D fortification of milk.

New research findings have documented fortification errors in fortified milk products all across the US milk industry. Also, the consumption of overfortified bovine milk has led the public to question whether vitamin fortification is safe. Therefore, North Carolina dairies were surveyed to determine vitamin fortification procedures used and to determine differences among these procedures. Of the parameters surveyed, the general conditions under which vitamin preparations were stored, the method used to add vitamin preparations to milk, and the point during processing at which vitamin preparations were added to milk were different among dairies. Forty-six percent of the dairies stored vitamin preparations under refrigerated conditions, and 54% stored vitamin preparations at ambient temperatures. The addition of vitamin preparations to bovine milk was accomplished by metered injection at 64% of the dairies and batch addition techniques at 36%. Vitamin preparations were added before fat content standardization and separation by 23% of the dairy processors; 77% added the vitamin preparations after this point. When dairies were asked at what point they added their vitamin preparations to the milk, nine different answers were given. Although other sources of error could also contribute to inconsistent concentrations of vitamin fortification, differences in fortification procedures may have a large impact upon the problem. The diversity of vitamin fortification practices used in North Carolina may be an indication of nationwide trends.

Animals↗
PubMed1994

Backbone dynamics of a two-domain protein: 15N relaxation studies of the amino-terminal fragment of urokinase-type plasminogen activator.

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) is a two-domain protein which consists of a kringle and a growth factor domain (GFD). The dynamics of uniformly 15N-labeled ATF was examined by measuring the longitudinal (T1) and transverse (T2) 15N relaxation times and heteronuclear NOEs. The data were interpreted in terms of the model-independent spectral density function. The GFD was found to exhibit a high degree of anisotropy, whereas the kringle domain of ATF undergoes isotropic reorientation. This difference in anisotropy is best explained by the two domains moving independently such as differently shaped beads on a string. With the exception of the N- and C-terminal regions of the protein, the most flexible region of ATF was the seven-residue omega loop (N22-I28) of the GFD which has been implicated in the binding of u-PA to its receptor. The amides of the linker region between the domains displayed high values of the order parameter, indicating restricted motion on the picosecond time scale. This is in contrast to the flexible linker of calmodulin [Barbato et al. (1992) Biochemistry 31, 5269-5278], which displayed low values of S2 and unrestricted motion in the linker region.

Kringles↗
PubMed1994

Solution structure of the amino-terminal fragment of urokinase-type plasminogen activator.

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator is a two domain protein which consists of a growth factor and a kringle domain. The 1H, 13C, and 15N chemical shifts of this protein have been assigned using heteronuclear two- and three-dimensional NMR experiments on selective and uniformly 15N- and 15N/13C-labeled protein isolated from mammalian cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data which resulted in a total of 1299 NOE restraints. The NOE restraints were used along with 27 phi angle restraints and 21 hydrogen-bonding restraints to produce 15 low energy structures. The individual domains in the structures are highly converged, but the two domains are structurally independent. The root mean square deviations (rmsd) between residues 11-46 in the growth factor domain and the mean atomic coordinates were 0.99 +/- 0.2 for backbone heavy atoms and 1.65 +/- 0.2 for all non-hydrogen atoms. For residues 55-130 in the kringle domain, the rmsd was 0.84 +/- 0.2 for backbone heavy atoms and 1.42 +/- 0.2 for all non-hydrogen atoms. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor and other homologous proteins were observed in the region which has been implicated in binding the urokinase receptor which may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

Amino Acid Sequence↗
PubMed1994

Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling.

A method is described for stereospecifically assigning the alpha-protons of glycine residues in proteins. The approach involves the stereospecific deuteration and 15N labeling of glycine and subsequent selective incorporation of this residue into the protein. The stereospecific assignments of the glycine alpha-protons are obtained from a comparison of a 3D 15N-resolved TOCSY spectrum of the uniformly 15N-labeled protein with a 2D/3D 15N-edited TOCSY spectrum of the protein, containing the stereospecifically deuterated and 15N-labeled glycine. The approach is demonstrated by stereospecifically assigning the glycine alpha-protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.

Carrier Proteins↗
PubMed1993

ENDOR and ESEEM studies of cytochrome c oxidase: evidence for exchangeable protons at the CuA site.

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used to study whether protons in the immediate protein environment around CuA in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the CuA site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 A between the CuA center and the protein/water interface. This relatively short surface separation distance is consistent with the role of CuA as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C. (1991) J. Biol. Chem. 266, 2219-2226).

Binding Sites↗
PubMed1992

A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells.

A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.

Amino Acids↗
PubMed1992

Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins.

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.

Affinity Labels↗
PubMed1992

Benzaldehyde, citral, and d-limonene flavor perception in the presence of casein and whey proteins.

The effect of sodium caseinate and whey protein concentrate on benzaldehyde, d-limonene, and citral flavor intensity was determined by quantitative descriptive analysis deviation from reference using a 12-member trained panel. The concentrations for the benzaldehyde, d-limonene, and citral flavor intensity references were 17.8, 53.0, and 19.8 ppm, respectively. The concentration for both protein references was .25%. Flavored protein solutions were held for 17 h at 6 degrees C and contained benzaldehyde (17.8 ppm), d-limonene (53 ppm), or citral (19.8 ppm) and 2.5% sucrose with 0, .125, .25, or .5% protein. Benzaldehyde flavor intensity declined as the whey protein concentrate concentration increased from 0 to .5%. There was no significant difference in benzaldehyde flavor intensity with casein compared with the reference. The d-limonene flavor intensity declined as the protein concentration increased. Panelists found no significant drop in citral flavor intensity with casein or whey protein. Decreased benzaldehyde and d-limonene flavor intensity in the presence of whey protein concentrate or casein may be due to nonpolar interactions (casein), interaction with nonpolar binding sites, cysteine-aldehyde condensation, or Schiff base formation (whey protein concentrate).

Acyclic Monoterpenes↗
PubMed1991

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.

Animals↗