Biosynthesis of neurophysin proteins in the dog and their isolation.

Biosynthesis of Neurophysin Proteins in the Dog and Their Isolation1 RODERICH WALTER, T. K. AUDHYA, DAVID H. SCHLESINGER, 2 S. SHIN,3 S. SAITO,4 AND H. SACHS4-5 Department of Physiology and Biophysics, University of Illinois at the Medical Center, Chicago, Illinois 60612 Ala-Met-Ser-Asp-Leu-Glu-Leu-. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vivo pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated. (Endocrinology 100: 162, 1977)

ABSTRACT. Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-LeuAsp - Leu - Asp - Val - Arg - Gin - Cys - Leu - Pro - Cys Gly-Pro-Gly-Gly-Gln-Gly- while that of dog Np- II is

P

HYSIOLOGICAL and morphological correlates provide evidence for the concept of neurosecretion advanced by Scharrer and colleagues (1,2), which proposes that neurosecretory neurons of the hypothalamo-neurohypophyseal complex synthesize, transport and secrete the neurohypophyseal hormones, oxytocin and vasopressin. During these processes the individual hormones are found in close association with a class of proteins referred to as neurophysins (3). Early in vivo pulse-chase experiments carReceived April 1, 1976. Biochemical Nomenclautre follows the recommendations of the IUPAC-IUB Commission,/ Biol Chem 247: 977, 1972. Additional abbreviations are: Np, Neurophysin; HME, hypothalamo-median eminence; TCA, trichloroacetic acid; EDTA, ethylenediaminete-traacetic acid. 1 Supported in part by the National Institute of Arthritis, Metabolism, and Digestive Diseases grant AM-18399. 2 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. 3 Present address: Department of Physiology, Faculty of Medicine, Queen's University, Kingston, Ontario, Canada. 4 Roche Institute of Molecular Biology, Nutley, New Jersey. 5 On leave of absence; present address: Case-Western Reserve University, School of Medicine, Department of Anatomy, Cleveland, Ohio.

ried out in the dog (4) and similar more recent studies using an organ culture of guinea pig hypothalamus (5) suggest that neurophysin biosynthesis involves the initial formation of a precursor molecule, as was originally reported for vasopressin biosynthesis (6,7). This manuscript describes experiments dealing with the isolation, characterization, and biosynthesis of neurophysin (Np) in the dog. In this study the initial labeling was performed in vivo for various time periods followed by removal of the hypothalamo-median eminence (HME) complex which was subsequently "chased" in vitro. Labeled Np and vasopressin were isolated simultaneously and the findings support the notion of the formation of precursor molecules from which the Np and the hormone are released. Furthermore, it is found that there are at least two different classes of Np molecules in the dog posterior pituitary and partial sequences of both have been established. Materials and Methods Radioactive amino acids For biosynthetic studies the preparation of [35S]cystine and its conversion to [35S]cysteine 162

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BIOSYNTHESIS OF NEUROSECRETORY MATERIAL prior to use was carried out as previously described (8). Tritium-labeled amino acids with specific activities of 20-40 Ci per mmol were purchased from New England Nuclear Corporation. Isotopic purity was checked by paper chromatography in appropriate solvent systems and the radiochromatograms were scanned with a Vanguard 880 automatic scanner. Immunological procedures a. Radioimmunoassay. Two different preparations of rabbit antisera to Np proteins were used as the experiments reported were performed over a period of a few years. In the initial labeling experiments described, the antibody preparation was obtained from rabbits inoculated with a mixture of bovine Nps. In more recent experiments rabbit antiporcine Np I serum (9), diluted 1:1000, was used for the radioimmunoassay of dog Nps. Both antibodies formed a precipitin line in the immunodiffusion technique (see below) with dog Nps. Radioimmunoassays were performed by the double antibody procedure (10). Purified Np from fresh dog pituitaries (dog NpII, see Results) was iodinated (2-3 ;u,g protein/mCi 125I) according to the method of Hunter and Greenwood (11), separated from free iodide on Sephadex G-25, and used in the competitive binding assays. Sheep anti-rabbit yglobulin (Gibco), diluted 1:5, was used to separate free from bound protein. b. Immunodiffusion. Ouchterlony double diffusion was carried out in 1% agarose made in 0.05M Tris-glycine-buffered 0.15M NaCl containing 0.01M EDTA and 0.1% sodium azide (pH 8.0) as supporting medium. Petri dishes (100 x 15 mm) containing 15 ml agarose were used, and a working arrangement consisted of wells 2.5 mm in diameter and spaced 4 mm apart. Np protein (15 /xg) was cross-reacted with 5 y\ of diluted antibody solution. The reaction was allowed to proceed at 35 C in a water-saturated incubator. Precipitation lines were observed after 48 h.

163

running pH of 8.4 at a current of 2.5 mA per gel (13). Protein was detected by staining with 0.05% (wt/vol) Coomassie brilliant blue in 12% TCA (14). After 1 h staining, the gel was destained for 24 h in 10% TCA. The gels containing labeled proteins were sectioned with the Joyce-Loeble slicer in one mm segments, which were homogenized in the HCl-acetic acid solution and after several hours at room temperature, the suspension was placed in a dioxane counting mixture (6) for determination of radioactivity in a liquid scintillation counter. Bioassays of neurophysins Oxytocin content at various steps in the preparation of Np was determined by the avian vasodepressor assay on conscious chickens (15) according to the procedure of Coon (16). Rat pressor activity was determined in atropinized, urethane-anesthetized male rats following the procedure in the U.S. Pharmacopeia (17). A match design was used for the bioassays using USP Posterior Pituitary Reference Standard as reference. Isolation of neurophysin proteins a. From fresh dog posterior pituitaries. Mongrel dogs, lightly anesthetized with sodium pentobarbital, were killed by an iv injection of about 40 ml of a saturated MgSO4 solution; the posterior pituitary glands were excised and immediately homogenized in 0.2M acetic acid—0.02M HC1 (10 ml per gland). The supernatant was separated by centrifugation at 20,000 x g for 30 min at 4 C. The supernatant was concentrated by lyophilization and subjected to gel filtration on Sephadex G-75 (0.9 x 33 cm) equilibrated with the acid solutions used for extraction of the tissues. The protein fractions were monitored by the procedure of Lowry et al. (12). The fractions emerging after 2 - 3 void volumes were pooled and lyophilized. The protein was then taken up in 2 ml of 0.05M acetic acid—0.823M

Polyacrylamide gel electrophoresis

pyridine buffer, pH 6.5, and chromatographed on DEAE Sephadex A-50 (0.9 x 20 cm) equilibrated with the same buffer. The Np was eluted by applying a linear gradient consisting of 50 ml each of 0.05M and 0.5M acetic acid. The pyridine concentration was 0.823M in both chambers and the pH of the buffers was 6.50 and 5.35, respectively.

Protein solution was applied on 7% gel (5.5 x 70 mm) or 15% gel (5.5 x 93 mm) with a

b. From frozen dog posterior pituitaries. Glands kept frozen at —196 C from the time of dissec-

Protein determination Protein concentration was determined according to the method of Lowry et al. (12).


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tion until processing were homogenized in a Potter Elvehjem type tissue grinder with O.lN HC1 (200 ml/86 posterior pituitaries) and stirred at 4 C for 20 h, and the insoluble materials were removed by centrifugation at 20,000 x g for 1 h in a Sorvall refrigerated centrifuge. After retaining the supernatant the residue was further homogenized with 100 ml of O.lN HC1 and was kept at 4 C while stirring. The supernatants were pooled, and the solution was adjusted to pH 6.8. The precipitate formed at neutral pH was removed by centrifugation at 4 C at 20,000 x g for 30 min. The supernatant was adjusted to pH 4.0 and 38 g NaCl (12.5% final concentration) was added very slowly with stirring at 4 C. Precipitation was allowed to continue for 20 h. The precipitate, which was removed by centrifugation at 24,000 x g for 1 h, was then dissolved in deionized water with a few drops of acetic acid and dialyzed for three days against distilled water (7 liters of water with six changes over a 3-day period) in a Spectrapor-3 membrane tubing. The retentate was lyophilized twice. Lyophilized protein was chromatographed on a Sephadex G-75 column (1.5 x 90 cm) and the crude dog Np was dissolved in 5 mM ammonium acetate buffer (3 ml), pH 5.0, and passed through a column of Cellex-CM (0.9 x 12 cm) equilibrated with the same buffer. The column was eluted with 20 mM ammonium acetate buffer (pH 6.9) and was run at a flow rate of 25 ml/h. The appropriate fractions were pooled and lyophilized. The protein, dissolved in 1 ml of 0.03M Tris-HCl buffer (pH 8.0), was applied to a Sephadex G-25 column (1.1 x 25 cm) and eluted with the same solvent. Two ml fractions were collected and the pooled fractions (6 ml) were lyophilized. The desalted Np protein was dissolved in 3 ml of deionized water and applied to a DEAE-Sephadex A-50 column (1.5 x 30 cm) equilibrated with 0.06M Tris-HCl buffer, pH 8.0. After the column had been charged with protein, it was washed with the same buffer for 6 h prior to elution with a continuous NaCl gradient and eluted at a flow rate of 8 ml/h with NaCl solution with an ionic strength gradient from 0.0-0.4M salt for a theoretical 430-ml volume. Further characterization of purified dog neurophysin proteins a. Microisoelectric focusing. Analytical isoelectrofocusing in polyacrylamide gels was per-

Endo < 1977 Vol 100 . No 1

formed according to the method of Karlson et al. (18) using a model LKB 2117 Multiphor isoelectrofocusing apparatus. b. Amino acid analysis. Polypeptides were hydrolyzed in 6N HC1 containing 0.05% (vol/ vol) /3-mercaptoethanol at 110 C in vacuo for 24 h. Amino acids were quantitated on a Model 121M Beckman amino acid analyzer by the method of Spackman et al. (19). Partial sequences of neurophysin I and 11 of dog For automated sequential analysis with a Beckman 890 protein sequencer using a double cleavage Quadrol program (20), samples of canine Np I and II were reduced with /3-mercaptoethanol and S-alkylated with [14C]iodoacetic acid (21) or oxidized with performic acid (22) (all reagents used in sequential analysis were purchased from Beckman Instruments). The thiazolinone resulting at each step of the degradation was converted to its isomeric phenylthiohydantoin (PTH) amino acid by heating in 0.2 ml of 1.0N HC1 at 80 C for 10 min (23). PTH amino acids residing in the ethyl acetate extract after conversion were identified by thinlayer chromatography using system Hl of Edman (24). Confirmation of these identifications and quantification of the PTH amino acids were made by gas chromatography (25) (Beckman GC 45 gas chromatograph) under isothermal conditions with Beckman SP400 as the solid-support resin. The polar PTH of arginine, which remained in the aqueous phase after conversion, was identified by thin-layer chromatography using a solvent composed of chloroform: methanol: heptafluorobutyric acid (26). After development of the thin-layer plate, the intensity of the PTH amino acids was enhanced by vapors of iodine (27). The plate was transilluminated by UV light at 254 nm and photographed for permanent records. The thin-layer plates were then subjected to autoradiographic development on X-ray film (28). Pulse-chase experiments These were carried out according to the design of the in vivo pulse—in vitro chase experiments previously described by Sachs and Takabatake (7). Either [35S]cysteine or one or more 3H-labeled amino acids were infused into the third ventricle of anesthetized dogs for 1.54.0 h, after which the HME complex was re-


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Quantification of labeled vasopressin and neurophysin-like proteins Unless otherwise stated HME tissues taken from dogs or incubation media were homogenized in 0.2M acetic acid—0.02N HC1 (5.0 ml/0.5 HME) and the insoluble materials were removed by centrifugation at 20,000 x g for 30 min in a Sorvall refrigerated centrifuge. Aliquots of the supernatant solution were removed for determination of protein, pressor activity and radioactivity. Arginine vasopressin (4 pressor units in rat, equivalent to ca. 10 yug) and purified Np (1.0 mg) were added as carrier and the solution was concentrated by lyophilization to 2-3 ml. Following the separation of proteins and vasopressin on a column of Sephadex G-25 (0.9 x 150 cm, equilibrated with 0.2N acetic acid— 0.02N HC1), the labeled hormone was isolated and purified to constant specific activity by means of procedures previously described (29). The protein fraction emerging in the void volume of the column was again concentrated to a small volume and passed consecutively through Sephadex G-50 (0.9 x 100 cm) and Sephadex G-75 (0.9 x 100 cm), each equilibrated with 0.2N acetic acid—0.02N HC1. Columns were run at a flow rate of 15-20 ml/h; the column effluents were collected in 2 ml fractions and each fraction was monitored for radioactivity and optical density at 280 nm. After gel filtration on Sephadex G-75, the fractions containing Np were combined, dialyzed against distilled water, and concentrated to a volume

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moved and divided in the medial plane. Onehalf of the HME was processed immediately as described below for the isolation of Np and vasopressin, while the other half was sliced (7) and incubated for 4.5 h at 37 C in a modified Krebs-Ringer solution (8) containing puromycin and the corresponding unlabeled amino acids prior to the isolation of labeled hormone and binding protein. In one experiment HME tissue was taken from a dog that had received an intraventricular infusion of [35S]cysteine 10 days prior to killing. This dog had been subjected to dehydration (5 days), intraventricular isotope infusion (6 h) and the rehydration (10 days) regimen previously described (7). The HME of this dog was treated as described above and, in addition, 35S-labeled Np and vasopressin were isolated separately form the neural lobe.

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FIG. 1. Isolation of dog Np from fresh posterior pituitary glands by DEAE-Sephadex A-50 column chromatography. The Np fraction from Sephadex G-75 (7 mg protein) was chromatographed on a column (1 x 31 cm) initially equilibrated with 0.1M acetic acid —0.82M pyridine, pH 6.1; elution was performed stepwise as described by Breslow et al. (31) with buffers containing 0.82M pyridine and increasing concentrations of acetic acid (0.1, 0.2, 0.3, 0.4, and 0.5M, respectively, indicated by the arrows). Aliquots (50 ix\) were taken for protein determinations by the method of Lowry et al. Fractions indicated by the shaded bars were pooled for further characterization.

of 3 - 5 ml. Aliquots were then taken for counting, protein determination, gel electrophoresis, chromatography on CM-Sephadex C-50 and immunodiffusion.

Results Antibodies raised in rabbits Analysis by means of double diffusion in agarose of antibodies raised either against a bovine Np mixture or against porcine Np I (30) showed a) precipitin bands to highly purified bovine Np I and II as well as to dog Np, b) a cross-reaction between bovine and dog Np, and c) lack of cross-reactivity between bovine Np and the higher molecular weight proteins present in acid extracts of bovine posterior pituitary. Isolation of neurophysin from fresh dog posterior pituitaries The acid extract of 9 posterior pituitaries taken from dogs within a few minutes after killing was first fractionated on Sephadex G-25 to separate proteins from


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FIG. 2. Isolation on a preparative scale of dog Np proteins from frozen posterior pituitaries by DEAE-Sephadex A-50 col02 umn chromatography. The desalted acidic protein (18 mg) from Sephadex G-25 column was chromatographed on a column (1.5 x 30 cm) previously equili40 80 120 160 200 240 280 320 360 400 440 brated with 0.06M Tris-HCl bufELUT ION VOLUME (ml) fer (pH 8.0). After the column was washed, the protein mixture was eluted in 4 ml fractions with the same buffer containing increasing concentrations of NaCl (0.0 to 0.4M over 420 ml). Fractions indicated by the shaded bars contained Np proteins as revealed by immunodiffusion. The proteins are referred to as dog Np I and II on the basis of their electrophoretic mobilities or polyacrylamide gel electrophoresis at a running pH of 8.4. The fractions containing protein I and II were pooled separately, dialyzed, lyophilized, and analyzed.

peptide hormones and other low molecular

for the isolation of bovine Nps (33). This

weight substances. The void volume fraction contained 10.5 mg of protein. Subsequent fractionation of this material on Sephadex G-75 resulted in two peaks at approximately equal intensity (measured at 280 nm) at fraction numbers 7 and 15, respectively. Fraction 12-17 (2-3 void volumes) contained material which showed immunological reactivity to a porcine Np antiserum, and these fractions were combined and chromatographed on DEAE Sephadex A-50 using a stepwise gradient elution procedure (31). The elution profile (Fig. 1) showed protein emerging early over a broad range (U), followed by a second fraction (dog Np II), and lastly, with continued elution, there appeared a smaller and evidently more acidic protein peak (dog Np I). The latter two protein fractions were characterized by radioimmunoassay for competitive binding with a porcine Np antiserum. The Nps were designated dog Np I and dog Np II on the basis of their electrophoretic mobilities on polyacrylamide gel, as has been done previously with Np-proteins isolated from other species (32).

increase of salt concentration increased the overall yield of precipitated protein by as much as 21% to give a yield of 52 mg of lyophilized protein. In view of the early work of Ginsburg and Ireland (34), it would appear that dog Np II is the protein preferentially precipitated at the higher salt concentration at pH 4. After column chromatography of the salt-precipitated protein on Sephadex G-75, which gave a similar elution profile as Np proteins purified from frozen bovine glands (Audhya and Walter, Submitted for publication), 23.1 mg of crude Np was obtained. This preparation had an avian vasodepressor activity of 0.12 U/mg. Following removal of neutral and basic proteins by cation exchange chromatography on Cellex-CM (yield 18.2 mg) and desalting by gelfiltrationon Sephadex G-25, acidic proteins were separated by chromatography on DEAE-Sephadex A-50. The first minor UV-absorbing peak eluted (Fig. 2) consisted of a number of unidentified acidic proteins as shown by acrylamide gel electrophoresis; these proteins did not cross-react with rabbit anti-porcine antibody. The second peak (elution volume 260-276 ml; molarity of the gradient at midpoint was 0.3M NaCl) and the third peak (356-436 ml; 0.38M NaCl) yielded 3.4 and 5.2 mg of protein, respectively. Both protein fractions gave sharp precipitin lines against rabbit anti-porcine antibody in a Ouchterlony

Isolation of canine neurophysins from frozen posterior pituitary The soluble proteins in the acid extract of dog posterior pituitary (86 glands) were precipitated with 12.5% NaCl instead of 10% as originally used by Hollenberg and Hope


BIOSYNTHESIS OF NEUROSECRETORY MATERIAL double diffusion system in 1% agarose, and are referred to as dog Np II (second peak, Fig. 3) and Np I (third peak) on the basis of their electrophoretic mobilities. Both preparations had no detectable avian vasodepressor activity and appeared homogeneous by analytical disc electrophoresis (Fig. 3). Two distinct bands, corresponding in their electrophoretic mobility to those shown in Fig. 3, were noted upon coelectrophoresis of the two proteins. In another study the aim was to isolate dog Np I only. For this purpose 15 mg acidic protein obtained from the Cellex-CM column, dissolved in 2 ml 0.06M Tris-HCl buffer, pH 8.0, was applied to the DEAESephadex column and was eluted with 0.2M NaCl in Tris-HCl buffer (120 ml). The column was then eluted with a gradient of sodium chloride (0.2-0.4M) in Tris-HCl buffer to a total volume of 100 ml. Dog Np I was eluted between 0.36M and 0.38M NaCl. This protein had similar gel electrophoretic patterns as that of other dog Np I obtained by gradient elution. Further characterization neurophysins

of purified dog

Proteins present in the dog Np I and II fractions possess acidic isoelectric points, as has been found for all Nps of other species. Dog Np II gave one band when subjected to isoelectric focusing between pH 3.5-9.5. The dog Np I fraction gave one major band (identified as [des-AlaVAla^Leu^Np I) along with two minor, very closely spaced diffuse bands (one of them identified as intact dog Np I). Not only the major band, but also the remaining minor band appears to be a metabolite of Np I, and not an unrelated protein impurity (see below). In accord with amino acid compositions of Nps from other species (31,35-37), for both protein fractions termed dog Np I and Np II, 14 cysteine, 1 tyrosine, and 3 phenylalanine residues per 10,000 molecular weight, as well as a high content of glycine, glutamic acid and proline were found. Notable differences between dog Np I and dog Np II

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FIG. 3. Analytical disc gel electrophoretic pattern of dog Np proteins from frozen glands, isolated by anionexchange chromatography. Protein concentration was 50 /xg/25 fA; running pH 8.4; staining dye Coomassie brilliant blue. Direction of protein migration is to the bottom of the gel (anode).

also follow similar differences in Np I and Np II of other species. For example, dog Np I contains histidine (a low value of 0.4 residue per 10,000 mol wt was obtained in three independent experiments) as was found for human, guinea pig and cow Nps (see Table 2 in ref. 32). On the other hand, dog Np II lacks histidine and contains one residue of methionine, as was found for human and cow Nps (see Table 2 in ref. 32). While dog Np I contains only one lysine residue, Np II has two lysine residues per 10,000 mol wt. Partial sequence of canine proteins

neurophysin

The N-terminal nineteen amino acid residues of dog Np I and seven residues of dog Np II were determined by the automated Edman degradation procedure (Scheme). The repetitive yield of PTH


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SCHEME: Partial amino acid sequence of dog neurophysins I and II and of dog [des-Ala-Ala-Leu]-NP I by the automated Edman degradation technique Canine neurophysin I (dog Np I): Canine [des-Ala'-Ala'-Leu'JNP I: Canine neurophysin II (dog Np II):

5 10 15 20 Ala-Ala-Leu-Asp-Leu-Asp- Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly- - -Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-GIy-Gln-GIy- ? -Cys-PheAla-Met-Ser-Asp-Leu-Glu-Leu-

amino acids at selected steps in separate automated Edman degradations of the proteins are shown in Fig. 4. The average yield of the degradation of dog Np II falls off more sharply than that of dog Np I (due to solvent extractive losses) since considerably less material was available for sequence analysis. Quantification of PTH amino acid recoveries at selected steps allowed identification of residues in the sequences of the major and minor components of dog Np I except at cycles 10, 12, 14 and 16 of Np I (the equivalent sequence positions in [des-Ala-Ala-Leu ]Np I are 13,15, 17 and 19) in which a coincident residue is found in both proteins. Identification and 200-i 100: IT)

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CYCLES OF DEGRADATION FIG. 4. Average yields of PTH amino acids at selected steps of the automated Edman degradation of S-alkylated dog neurophysin proteins. Intact dog Np I (a minor component of the dog Np I fraction) (x x), a metabolite of dog Np I, [des-Ala'-Ala^Leu^Np I (the major component of the dog Np I fraction) (O O), and dog Np II ( • •).

quantification of phenylalanine at cycle 19 of the sequence of the major component of dog Np I extends knowledge of the intact dog Np I sequence through position 22, but we report only the contiguous sequence through position 19 in native dog Np I. The N-terminal heptapeptide sequence of dog Np II is identical to that earlier obtained with porcine I (35), bovine Np II (38) and ovine NP III (39). Dog Np I and II both possess an N-terminal alanine residue as has been found so far for all "native" Np proteins, but the glutamine residue found in position 18 is to date unique to dog Np I; it is expected on the basis of amino acid composition that dog Np II, which possesses one additional lysine, contains this basic residue in position 18. One of the two metabolites detected by isoelectric focusing in the dog Np I preparation possesses the same amino acid sequence as dog Np I, but lacks the N-terminal sequence Ala-Ala-Leu; this finding is reminiscent of that of Breslow et al. (31) who isolated bovine Np II from which the aminoterminal Ala-Met peptide had been removed by proteolytic digestion. Nevertheless, the removal of the amino-terminal tripeptide sequence from dog Np I, which did not contain any residues with formal charges, does not explain the separation by isoelectric focusing; enzymatic removal of a portion of the Np sequence from the C-terminus may account for our results. In order to explain our sequence data, we further have to assume that the second contaminant present in the dog Np I preparation has the intact amino-terminal sequence of dog Np I, but is partially digested at the C-terminus. This assumption also would explain our finding of a low histidine content in the amino acid composition of the dog Np I fraction. It


169

BIOSYNTHESIS OF NEUROSECRETORY MATERIAL may be deduced from the amino acid sequence of porcine Np I (35) that the location of histidine in Np proteins is in position 86; this position is only 7 residues from the C-terminus of the protein. In view of the amino acid composition, it is unlikely that a completely unrelated protein with a blocked N-terminus was the contaminant. Incorporation studies and identification of labeled neurosecretory material The design of the in vivo pulse—in vitro chase experiments has been described in Materials and Methods. The Np-like proteins constitute between 15 to 21% of the starting acid insoluble protein before the chase, and between 29-33% after the chase. After successive gel filtrations of the acid extracts of the HME segments, the labeled proteins in the Np region of the eluate were chromatographed on CM-Sephadex C-50 and the results are shown in Fig. 5. Radioactive proteins were found in the unretarded fraction in a subsequent well defined peak of counts, and this was followed by a smaller more diffuse peak of 35Slabeled protein; the latter two fractions (marked by arrows) corresponded to the position of elution for bovine Np I and Np II and were the only ones which reacted with a rabbit anti-bovine Np serum. As can be seen, the first Np fraction obtained from the HME portion which had been incubated in vitro (bottom half, Fig. 5) contained considerably more counts than that of the corresponding half of HME extracted immediately after the 1.5 h in vivo labeling period. Polyacrylamide gel electrophoresis of this fraction from the two HME halves gave the labeling patterns shown in Fig. 6; the radioactive material from the HME segment "chased" in vitro migrated directly behind the dye front and showed a well defined peak of counts which was several times greater than the same fraction obtained from the unincubated HME half. In the presence of anti-porcine Np sera, these same fractions formed antigen-antibody complexes, most of which were soluble

1000 800 600 400

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FIG. 5. Biosynthesis and turnover of Np-like proteins studied by an "in vivo pulse—in vitro chase" experiment. Following a 1.5 h pulse in vivo with [35S]cysteine and a 4 h chase in vitro, the pituitary was isolated and divided in half. Both pituitary halves were processed separately. The results of the in vivo pulse are shown above and of the in vitro chase below. Labeled tissue extracts were first passed consecutively through Sephadex G-25, G-50, G-75 and the Np containing fractions from the latter columns were pooled, lyophilized, and chromatographed on CMSephadex C-50 (0.9 x 20 cm) by means of the gradient elution procedure described by Hollenberg et al. (33); the flow rate was 10 ml/h and the fraction size was 1.5 ml. Only fractions marked by arrows reacted with a rabbit antibovine Np serum.

(80-85%) and could be detected in the void volume after gel filtration on Sephadex G-100 or G-75. Estimates of the total counts in Np-like protein from the HME extracts following gel filtration on Sephadex G-75 were obtained by this procedure after correction for non-specific binding (5-20%) observed in duplicate analysis with normal rabbit serum or in the presence of excess bovine Np. Between 17 to 22% of the total


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bonded radioactive peptides or amino acids, did not alter the counts in the material from the "chased" HME tissue, whereas a 200 considerable number of counts were removed from the labeled material derived from the unincubated portion of the HME. 150 On Sephadex G-50 columns (0.9 x 110 cm, Q. O 0.05M Tris-HCl, 0. 1M KC1, pH 7.5) calibrated with proteins of known molecular size, 100 labeled dog Np emerged slightly after cytochrome C, and close to bovine Np, and was estimated to have a molecular weight of about 11,000-12,000. In the same experiment, 35S-labeled vasopressin was purified to constant specific activity by procedures previously described FIG. 6. Polyacrylamide gel electrophoresis of labeled dog Np I. Aliquots of the first Np fraction emerg- for guinea-pig vasopressin (8), and the reing from the CM-Sephadex C-50 column (Fig. 6) were sults showed that the total number of counts electrophoresed on 7.5% gels according to Davis et al. of hormone also increased after the in vitro (13). Radioactivity in gel slices was counted as dechase (Table 1). In fact, this parallelism scribed in Materials and Methods; (O O) HME half pulsed in vivo 1.5 h; ( • — • ) HME half "chased" between vasopressin and Np labeling was observed in all of the experiments perin vitro. Arrow indicates the direction of migration. formed. The recovery of vasopressin and Npradioactivity of Sephadex G-75 column ef- like protein from the two HME halves was fluents was Np-like protein before the about the same. On the other hand, the chase, whereas the similar fraction after specific activity (cpm//u,g) of the proteins the chase gave a yield between 38-45%. insoluble in the acid extraction medium Treatment of these antigen-antibody com- either decreased or increased slightly during plexes with a solution of sodium sulfite and the in vitro incubation (Table 1). We do sodium tetrathionate in 6M guanidine HC1 not consider these differences in the amount followed by dialysis, in order to remove S-S of the acid insoluble protein as significant, 250

TABLE 1. Labeling of neurophysin and vasopressin of the dog after an in vivo pulse and in vitro chase Half HME, pulse in vivo

Exp. no.

Infusion time1 (h)

1 2 3 4

1.5 1.5 3.0 3.0 6.0

5*

Neurophysinlike proteins Total counts2 8,500 1,800 1,000 6,000 9,200

Half HME, chase in vitro

Purified 1 vasopressin Acidinsol. proPressor3 Total counts4 tein5 0.7 3.3 2.6 2.2 0.6

1,188 0 891 830 979

41 146 43 120

Neurophysinlike proteins Total counts2 19,200 4,400 1,300 18,200 31,000

Purifiec1 vasopressin Acidinsol. Presprosor3 'Total counts4 tein5 0.6 2.2 2.0 2.2 0.7

6,105 765 1,584 1,288 5,638

60 93 53 60

1 35

[ S]cysteine, 1.3-2.0 x 109 cpm, infused into the third ventricle at a rate of about 2.5 ml/h. Estimate obtained from counts in antigen-antibody complexes obtained from G-75 neurophysin fractions treated with beef neurophysin antisera. 3 Rat pressor activity expressed in units/mg protein. 4 Counts were obtained after purification according to Takabatake and Sachs (8). 5 Amount of acid insoluble proteins is expressed in cpm//u,g. * Killed 10 days later; see Materials and Methods. 2


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BIOSYNTHESIS OF NEUROSECRETORY MATERIAL since they are only a small fraction of the amount of label incorporated into Np and vasopressin. It shows the variabilities in experimental techniques, but still a consistent and high increase in Np-like protein and vasopressin is evident after the chase. In experiment no. 5, Table 1, a dehydrated dog had received an intraventricular infusion of [35S]cysteine for a period of 6 h and was then permitted to survive for an additional 10 days with free access to water before killing. Surprisingly, the HME tissue of this dog still produced [35S] vasopressin and [35S]neurophysin upon incubation in vitro, whereas under the same conditions the specific activity of the acid-insoluble protein decreased. In this experiment the neural lobe contained about 6.1 U of vasopressin. Evidently during the 10 day rehydration period, the dog had not yet replenished its full complement of posterior pituitary hormone which contained about 20,000 cpm after precipitation of tissue extract with TCA. In another experiment (not shown in Table 1), the extraction of the HME tissues was done with 0.1M pyridine acetate buffer, pH 5.8, containing 0.05% Triton X-100, instead of the HCl-acetic acid mixture, in order to examine a greater range of the Nplike and other proteins labeled during the initial in vivo pulse and after the in vitro chase. In this case the initial intraventricular infusion was for a period of 4 h with a mixture of 3H-labeled amino acids (Leu, Tyr, and Pro) rather than with [35S]cysteine. Following gel filtration on Sephadex G-25 and G-50 in the same buffer system as employed previously, the Np-containing fraction was subjected to electrophoresis on 15% polyacrylamide gels; the results are seen in Fig. 7. While after the in vivo infusion the dog Np peak is rather diffuse with low counts (upper panel, Fig. 7), the material from the incubated ("chased") portion of the HME showed a well defined peak (lower panel, Fig. 7) with the mobility of dog Np and the counts here were considerably greater. In the chased portion of the HME,

300

20

30

40

60

GEL SLICE NUMBER

FIG. 7. Biosynthesis and turnover of Np-like proteins studied by an "in vivo pulse—in vivo chase" experiment. Polyacrylamide gel electrophoresis of 3Hlabeled proteins from dog HME halves after 4 h pulse in vivo (3H-labeled Leu, Tyr and Pro) (upper panel), followed by a 4 h "chase" in vitro (lower panel). Unlike in other experiments with labeled Np, in this experiment the two HME halves were extracted with O.lM pyridine acetate, pH 5.8 containing 0.05% Triton X-100 and the extracts were chromatographed on Sephadex G-25, and then on Sephadex G-75. The Np fraction from the latter column was electrophoresed on 15% gels, the gels were stained with Coomassie blue, and gel slices were dissolved in 30% H2O2 prior to counting; solid line, optical density, (• • ) 3H-counts per one mm gel slice.

a diminution of labeled protein at the top of the gel was also observed along with an enhanced labeling of at least 2 additional protein bands of unknown character with mobilities less than that of dog Np. Discussion Neurosecretory granules isolated from posterior pituitaries of various species have been found to contain not only the neurohypophyseal hormones but Np proteins as well (33,40). Nps are a family of low molecular weight proteins with surprisingly


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similar primary structures (41-43). Available evidence indicates that oxytocin and vasopressin are produced in different neurons and are each associated with a separate Np (44-47). Fractionation of acid extracts of fresh or frozen dog posterior pituitaries have likewise indicated the presence of at least two Np proteins. In our initial experiments we used fresh dog pituitaries and the isolation followed standard procedures (31,33) with minor modification. For the isolation of dog neurophysin proteins on a preparative scale, we started with frozen pituitaries and followed an improved purification scheme recently developed for the isolation of bovine Nps, in which the acidic Np proteins are separated from the large amounts of neutral and basic components by cation-exchange chromatography (Audhya and Walter, Submitted for publication). The two dog Nps isolated were characterized by analytical gel electrophoresis, immunological studies, amino acid composition and partial sequence determination. The first seven residues of dog Np II have been determined and the sequence is identical to that found in bovine II, porcine Np I and ovine Np III (35,38,39). Dog Np I possesses an N-terminal Ala-Ala sequence as was found previously for human Np I (36,48). Recently Acher and colleagues (49) suggested the existence on the basis of sequence data of two classes of Nps (MSELand VLDV-Np according to the amino acids in positions 2, 3, 6, and 7 of the proteins). However, the dog and human Np I are not compatible with either group, which shows that in view of the limited sequence data available to date it may be premature to advance such a general hypothesis. While the dog Np II preparation was of high purity, the dog Np I preparation contained at least two contaminants which appear to be catabolites of "native" dog Np I. Our results raise the possibility that partial enzymatic degradation of the "native" Nps may not only occur during isolation using a dilute H2SO4 extraction procedure (31,46),

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1977 No 1

but that these metabolites may already be present in vivo, since the HC1 extraction method has been found to inhibit the cathepsin-like enzymes thought to be responsible for the digestion of Nps (33,46). The recent finding by Vilhardt and Robinson (50) of the presence of minor Np components in neurosecretory granules prepared from fresh tissue of cow might also indicate the "in vivo" formation of bovine Np metabolites. Previous work on vasopressin biosynthesis (7,8,29) has provided evidence indicating the involvement of a precursor molecule whose formation utilizes the cellular protein synthetic machinery. The combined results of analytic studies on normal, dehydrated, and water repleted rats and on the Brattelboro strain of rats with diabetes insipidus, as well as isotope incorporation experiments in the dog, suggest that the biosynthesis of the hormone and of Np are closely related events (51-54). In light of the foregoing, the existence of a shared precursor is not only an interesting possibility but would also constitute a convenient mechanism for the coordinated biosynthesis and packaging of these two neurosecretory components. However, experimental results to date neither show that the precursors of vasopressin and Np are one and the same molecule nor do they rule out the existence of two different precursors with a biosynthetic pathway under common genetic control. The studies reported in this paper have attempted to examine further the possible involvement of a precursor in Np biosynthesis. The approach to this problem has been to label the putative precursor in vivo, remove the hypothalamic tissue from the animal and to "chase" in vitro under conditions which preclude de novo peptide bond synthesis (i.e., in the presence of inhibitors of protein synthesis). The results have shown that the in vitro "chase" invariably leads to the appearance of radioactive Np-like protein and vasopressin, both arising from material(s) which presumably


BIOSYNTHESIS OF NEUROSECRETORY MATERIAL must have been labeled during the initial in vivo pulse. In theory, we might have expected to see a ratio of Np-like protein to vasopressin of 7.0, since the number of cysteine residues in dog Nps, similar to Nps of other species, is 14 and that in vasopressin is 2. But the average ratio of the ten experiments (in vivo and in vitro) was 5.4 ± 4.3. Considering the limitations of the experiments with different dogs, such a high standard deviation may be acceptable. Analogous observations have been made in studies on Np biosynthesis in guinea pig hypothalamic organ culture (5). It is also interesting to note that no increase of pressor activity was detected when comparing the appropriate values of the in vivo pulse and in vitro chase data. This result may indicate that there is no increase in total vasopressin content, but rather only in specific radioactivity since the rate of hormone biosynthesis and degradation may have reached an equilibrium. A similar argument could account for the corresponding Np data. Experiment No. 5, Table 1, in which a dog was killed 10 days after isotope infusion and in which the "chase" in vitro still gave rise to radioactive hormone and Np, requires some comment. Here it was expected that labeled precursor(s) would have been processed within the tenday interval (i.e., before the "chase"). However, in the case of the rat, it was observed that rehydration led to a progressive reaccumulation of these secretory materials in the neural lobes and that an approximately normal amount was only found in animals killed 14 days after rehydration (55). Therefore, the ten-day interval may not have been sufficiently long to replenish vasopressin and Np-like material in the dog. Another, and maybe more likely, explanation of our observations could be reutilization of isotope due to protein turnover in vivo. The findings reported here also confirm previous studies on vasopressin biosynthesis in the dog (8) and in the guinea pig (5), and furthermore lend support to

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the postulate of the existence of a Np precursor. The available data, however, do not distinguish between the alternatives of either a shared or a separate precursor for the hormone and Np, and in fact, ultimate proof of the above postulate must depend upon the isolation and characterization of the precursor(s). Acknowledgments The authors wish to thank Drs. Henry Friesen and K. W. Cheng of the University of Manitoba, Winnipeg, Canada, for supplying us with antiporcine antibodies. We are also thankful to Ms. Alice Formento for performing bioassays and to Mr. Michael Ernst for excellent technical assistance.

References 1. Bargmann, W., and E. Scharrer, Am Scientist 39: 255, 1951. 2. Scharrer, E., and B. Scharrer, Recent Prog Horm Res 10: 183, 1954. 3. Acher, R., G. Manoussos, and G. Olivry, Biochim Biophys Ada 16: 155, 1955. 4. Fawcett, C. P., A. Powell, and H. Sachs, Endocrinology 83: 1299, 1968. 5. Sachs, H., D. Pearson, and A. Nureddin, Ann NY Acad Sci 248: 36, 1975. 6. Sachs, H . J Neurochem 10: 299, 1963. 7. Sachs, H., and Y. Takabatake, Endocrinology 75: 943, 1964. 8. Takabatake, Y., and H. Sachs, Endocrinology 75: 934, 1964. 9. Cheng, K. W., and H. G. Friesen, Endocrinology 88: 608, 1971. 10. Legros, J. J., P. Franchimont, and J. C. Hendrick, C R Soc Biol (Paris) 163: 2773, 1969. 11. Hunter, W. M., and F. C. Greenwood, Nature (London) 194: 495, 1962. 12. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 13. Davis, B. J., Ann NY Acad Sci 121: 404, 1964. 14. Chrambach, A., R. A. Reisfeld, W. Wyckoff, and J. Zaccari, Anal Biochem 20: 150, 1967. 15. Munsick, R. A., W. H. Sawyer, and H. B. Van Dyke, Endocrinology 66: 860, 1960. 16. Coon, J. M., Arch lnt Pharmacodyn Thre 62: 79, 1939. 17. The Pharmacopeia of the United States of America, 18th Revision, Mack Publishing, Easton, Pa., 1970, p. 464. 18. Karlson, C , H. Davies, and U. B. Anderson, LKB 2117 Multiphor Application Note-I, 1973. 19. Spackman, D. H., W. H. Stein, and S. Moore, Anal Chem 30: 1190, 1958.


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20. Edman, P., and G. Begg, Eur J Biochem 1: 80, 1967. 21. Anfinsen, C. B., and E. Haber, J Biol Chem 236: 1361, 1961. 22. Schlesinger, D. H., H. D. Niall, and D. Wilson, Biochem Biophys Res Commun 61: 282, 1974. 23. Niall, H. D., In Hirs, C. H. W., and S. Timasheff (eds.), Methods in Enzymology, Academic Press, New York, 1973, p. 942. 24. Edman, P., In Needleman, S. (ed.), Protein Sequence Determination, Springer-Verlag, New York, 1970, p. 211. 25. Pizano, J. J., and T. J. Bronzert,/ Biol Chem 244: 5597, 1969. 26. Schlesinger, D. H., G. Goldstein, and H. D. Niall, Biochemistry 14: 2214, 1975. 27. Ingles, A. S., P. W. Nicholls, and L. G. Sparrow, J Chromatogr 90: 362, 1974. 28. Jacobs, J. W., R. T. Sauer, H. D. Niall, H. T. Keutman, J. L. H. O'Riordan, G. P. Aurbach, and J. T. Potts, Jr., Fed Proc 32: 668, 1973. 29. Sachs, H . J Neurochem 5: 297, 1960. 30. Cheng, K. W., and H. G. Friesen, Endocrinology 88: 608, 1971. 31. Breslow, E., H. L. Aanning, L. Abrash, and M. SchmirJ Biol Chem 246: 5179, 1971. 32. Walter, R., and E. Breslow, In Marks, N., and R. Rodnight (eds.), Research Methods in Neurochemistry, vol. 2, Plenum Press, New York, 1974, p. 247. 33. Hollenberg, M. D., and D. B. Hope, Biochem J 106: 557, 1968. 34. Ginsburg, M., and M. J. Ireland, J Endocrinol 30: 131, 1964. 35. Wuu, T. C., S. Crumm, and M. Saffran, / Biol Chem 246: 6043, 1971. 36. Cheng, K. W., and H. G. Friesen, / Clin Endocrinol Metab 34: 165, 1972. 37. Uttenthal, L. O., and D. B. Hope, Biochem J 116: 899, 1970.

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38. Schlesinger, D. H., B. Frangione, and R. Walter, Proc Natl Acad Sci USA 69: 3350, 1972. 39. Schlesinger, D. H., M. Ernst, A. Nicholas, W. B. Watkins, and R. Walter, FEBS Letters 57: 55, 1975. 40. Dean, C. R., and D. B. Hope, Biochem J 106: 565, 1968. 41. Capra, J. D., and R. Walter, Ann NY Acad Sci 248: 397, 1975. 42. Chauvet, M. T., J. Chauvet, and R. Acher, FEBS Letters 58: 234, 1975. 43. Walter, R., D. H. Schlesinger, I. H. Schwartz, and J. D. Capra, Biochim Biophys Res Commun 44: 293, 1971. 44. Dyball, R. E. J., and M. J. Brimble, Fifth International Congress of Endocrinology, Hamburg, 1976, p. 198 (Abstract). 45. Sokol, H. W., E. A. Zimmerman, W. H. Sawyer, and A. G. Robinson, Endocrinology 98: 1176, 1976. 46. Dean, C. R., D. B. Hope, and T. Kazic, Br J Pharmacol 34: 1928, 1968. 47. Pickup, J. T., C. I. Johnston, S. Nakam, L. O. Uttenthal, and D. B. Hope, Biochem J 132: 361, 1973. 48. Capra, J. D., K. W. Cheng, H. G. Friesen, W. G. North, and R. Walter, FEBS Letters 46: 71, 1974. 49. Chauvet, M. T., J. Chauvet, and R. Acher, FEBS Letters 52: 212, 1975. 50. Vilhardt, H., and I. C. A. F. Robinson, / Neurochem 24: 1275, 1975. 51. Valtin, H., W. H. Sawyer, and H. W. Sokol, Endocrinology 77: 701, 1965. 52. Rennels, M. L., Endocrinology 78: 659, 1966. 53. Friesen, H., and E. B. Astwod, Endocrinology 80: 278, 1967. 54. Burford, G. D., C. W. Jones, and B. T. Pickering, Biochem J 124: 809, 1971. 55. Young, T. K., and H. B. Van Dyke, / Endocrinol 40: 337, 1968.

International Society of Psychoneuroendocrinology Preliminary Notice. The Eighth International Congress of the International Society of Psychoneuroendocrinology will be held at the Atlanta Hilton Hotel, Atlanta, Georgia, May 8-12, 1977. Registration will be on Sunday, May 8. For information please write to Dr. Richard P. Michael, Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia 30322.


Neurophysin biosynthesis: conversion of a putative precursor during axonal transport.

Isolation in high yield of separated rat neurophysin-I and neurophysin-II by combined cation and anion exchange chromatography.

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis.

Characterization of goat-testes antigens and effect of their immunization on biosynthesis of proteins in testes.

Non-histone chromosomal proteins. Their isolation and role in determining specificity of transcription in vitro.

Isolation of mutants blocked in calicheamicin biosynthesis.

The complete amino acid sequence of the major ovine neurophysin (MSEL-neurophysin); comparison with a re-investigated bovine MSEL-neurophysin.

Isolation and some properties of dog and rat pancreatic kallikreins.

Terpenoids and their biosynthesis in cyanobacteria.

Isolation of peroxisomes from the dog kidney cortex.

"She's a dog at the end of the day": Guide dog owners' perspectives on the behaviour of their guide dog.

Isolation and characterization of insoluble collagen of dog hearts.

Rapid isolation of specific DNA-binding proteins and their DNA-binding domains.

Identification, in the external region of the rat median eminence, of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres.

Isolation of luminal and antiluminal membranes from dog kidney cortex.

Phosphorylation of ribosomal proteins and the inhibition of protein biosynthesis.

Isolation, structural characterization and pharmacological activity of dog neuromedin U.

The serine proteinase inhibitory proteins of the human intervertebral disc: their isolation, characterization and variation with ageing and degeneration.

The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

Isolation of Chlamydia psittaci from pleural effusion in a dog.

Two separate differentiation inducing proteins for human myeloid leukemia cells and their isolation from normal lymphocytes.

Isolation and identification of local Bacillus isolates for xylanase biosynthesis.

Isolation and Characterization of O-methyltransferases Involved in the Biosynthesis of Glaucine in Glaucium flavum.

Somatostatin neurons and their projections in dog diencephalon.