Multiple molecular forms of phosphoprotein phosphatase. Separation of four forms of the rabbit skeletal muscle enzyme.

Phosphoprotein phosphatase [phosphoprotein phosphohydrolase EC 3.1.3.16] in the soluble fraction of rabbit skeletal muscle, when assayed with phosphorylase a[EC 2.4.1.1] from rabbit skeletal muscle and phosphohistone as substrates, was resolved into three active fractions (Fractions I, II, and III in order of elution) by DEAE-cellulose column chromatography. Sucrose density gradient centrifugation showed that these fractions were composed of subfractions of different molecular size (I: 7.3S and 4S; II: 8S and 4S; III; 6.7S). Components with larger molecular size in the major fractions, II and III, were dissociated to a molecular size similar to that of the smallest component on freezing in the presence of mercaptoethanol. These results indicate that phosphoprotein phosphatase from skeletal muscle occurs in multiple forms very similar to those of the liver enzyme reported previously (Kobayashi, Kato and Sato (1975) Biochim. Biophys. Acta. 373, 343-355).     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.     

Human beta-endorphin. Synthesis and biological activity of analogs with modification in residue positions 8, 27, and 9 or 11

Three analogs of human beta-endorphin (beta h-ER) were synthesized by the solid-phase method: [Gln8,Trp27]-beta h-EP (I), [Gln8,Arg9,Trp27]-beta h-EP (II), and [Gln9,Arg11,Trp27]-beta h-EP (III). Radioreceptor binding assay with use of tritiated beta h-EP as primary ligand gave relative potencies as follows: beta h-EP, 100;I, 778;II, 467;III, 449. Relative potencies in an analgesic assay were: beta h-EP, 100;I, 114;II, 165;III, 83. The 8-11 segment of beta h-EP can tolerate a net increase in charge of +2 without diminishing analgesic potency. The substitution of Glu8 may be one of the more dependable means of designing beta-endorphin antagonists.     

Separation of the alpha chains of type I and III collagens by SDS-polyacrylamide gel electrophoresis.

A method for the separation of type III collagen from type I collagen by SDS-polyacrylamide gel electrophoresis has been developed. This is based on the observation that the presence of 3-4 M urea decreases the mobility of the alpha 1 [III] chain to a greater extent than those of the alpha 1[I] and alpha 2 chains, although the alpha 1[I] and alpha 1[III] chains move at the same rate in the absence of urea. An attempt to separate the alpha 1[II] chain of type II collagen from the alpha 1[I] chain was unsuccessful under the experimental conditions employed.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis)

42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+.     

Studies on the metabolic inversion of the 6'chiral center of simvastatin.

In two simvastatin (SV) metabolites the 6' alpha-methyl of SV is oxidized to either 6' beta-CH2OH (I) or 6' beta-COOH (II). A possible intermediate is 6' exomethylene SV (III). When Sprague Dawley rats received an i.v. dose of [14C] III (1 mg/kg) metabolite II was excreted in bile. When dogs received an i.v. dose of [14C] III together with either [3H] SV (1 mg/kg) or its hydroxy acid form, [( 3H] SVA) (10 mg/kg), both 3H and 14C I and II were excreted in bile. These results strongly indicate that I and II are secondary metabolites of SV formed from III perhaps via a common aldehyde intermediate.     

Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins

The low molecular weight NADH dehydrogenase which can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents consists of three subunits in equimolar ratio [Galante, Y. M., & Hatefi, Y. (1979) Arch. Biochem. Biophys. 192, 559]. The largest subunit (subunit I) can be completely separated from the other two (subunits II + III) by treatment with sodium trichloroacetate and ammonium sulfate fractionation. Both the subunit I and subunit II + III fractions contain iron and acid-labile sulfur. From visible and EPR spectroscopy and the iron and acid-labile sulfide content, we propose that the subunit II + III fraction contains a binuclear cluster. The cluster structure present in subunit I is as yet unclear. On separation of the subunits of NADH dehydrogenase, the FMN is lost.     

Enzymatic formation of angiotensins II and III in the hindlimb circulation of dogs

Experiments were performed in 14 anesthetized dogs to (1) to determine if the reductions in hindlimb blood flow produced by [des-Asp1] angiotensin I were due to its local enzymatic (kininase II) conversion to angiotensin III and (2) to quantitate the extent of conversion of angiotensin I to angiotensin II and of [des-Asp1] angiotensin I to angiotensin III in the hindlimb circulation. Graded doses of these peptides were administered as bolus injections directly into the left external iliac artery while measuring flow in this artery electromagnetically. Dose-response relationships were determined before and during the inhibition of kininase II activity with captopril or antagonism of angiotensin receptor sites with [Ile7] angiotensin III. Captopril inhibited the vasoconstrictor responses to angiotensin I and [des-Asp1] angiotensin I, but did not affect the responses to angiotensins II or III, or norepinephrine. [Ile7] angiotensin III inhibited the vasoconstrictor responses to all four angiotensin peptides but did not alter the responses to norepinephrine. These findings indicate that the hindlimb vasoconstrictor responses to [des-Asp1] angiotensin I were due to the local formation of angiotensin III. The extent of conversion of [des-Asp1] angiotensin I to angiotensin III that occurred in one transit through the hindlimb arterial circulation was estimated to be 36.7%, which was not different from the estimated 36.4% conversion of angiotensin I to angiotensin II. We conclude that angiotensin I and [des-Asp1] angiotensin I are converted to their respective vasoactive forms (angiotensins II and III) to a similar extent in the hindlimb circulation via the action of kininase II.     

Analysis of 13NH4+ Efflux in Spruce Roots (A Test Case for Phase Identification in Compartmental Analysis)

13NH4+-efflux analyses were conducted with roots of intact Picea glauca (Moench) Voss. seedlings at external NH4+ concentrations of 100 [mu]M and 1.5 mM. Three kinetically distinct phases were identified with half-lives of exchange of approximately 2 s, 30 s, and 14 min. The presumed identities of the subcellular compartments corresponding to these phases were confirmed by several techniques, including pretreatment of roots (a) at 75[deg]C or with SDS, (b) with [alpha]-keto-glutarate or L-methionine-DL-sulfoximine, (c) at elevated levels of Ca2+, and (d) at low pH or with Al3+ at low pH. Treatments a and b selectively influenced phase III without affecting phases I and II. Similarly, treatment c selectively perturbed phase II, and treatment d affected phases II and III. Based on these findings and the assumption of an in-series arrangement of root cell compartments, it was concluded that phase III corresponded to the cytoplasm, phase II corresponded to the Donnan free space, and phase I corresponded to a film of solution adhering to the root surface.     

Palladium(II) salt and complexes of spermidine with a six-member chelate ring. Synthesis, characterization, and initial DNA-binding and antitumor studies.

By reaction of spermidine trihydrochloride with K2PdCl4 and PdCl2 at different pH's, we have synthesized the [sperH3]2[PdCl4]3 (I), [PdCl2(sperH)]2[PdCl4] (II), and [(PdCl2)3(sper)2] (III) compounds. The structure of these compounds was studied by IR and 1H NMR; complex II was analyzed by x-ray diffraction. In this complex the spermidine is attached to the PdCl2 group forming a six-member chelate ring with a protonated terminal amine group. The crystal of [PdCl2(sperH)]2[PdCl4] x 2H2O (II) is monoclinic, P2(1)/n, with a = 7.023(1) A, b = 12.662(1) A, c = 18.435(3) A, and beta = 99.95(1) degrees, Z = 4, R = 0.051, and Rw = 0.058 on the basis of 2690 independent reflections. We have compared the antitumor activity in vitro against the isolated human breast carcinoma MDA-MB 468 cell line of compounds I, II, and III with that of cis-diamminedichloroplatinum(II), cis-DDP. The results show that compounds III and III have values of ID50 similar (0.74 microgram/ml) or even lower (0.56 microgram/ml) than cis-DDP (0.80 microgram/ml). We also observed that compounds I, II, and III have the ability to induce conformational changes in covalently closed circular (ccc) form of the pUC8 plasmid DNA. Compounds II and III also induce conformational changes in the open circular (oc) form of this plasmid.     

Cholinergic Binding to the Receptor Proteolipid from Torpedo Electroplax Separated by Ion Exchange Chromatography

Abstract

Proteolipids (i.e., hydrophobic proteins) have been extracted with chloroform-methanol (2:1) from lyophilized Torpedo electroplax, and fractionated on a DEAE-cellulose column. The elution system consisted of the same solvent and a gradient of the hydrophobic ion ptoluene sulfonate (0.1–100mM). The three fractions obtained (I, II and III) have different content of protein, lipid P, and reducing sugars. The amino acid composition shows a higher proportion of hydrophobic residues in I and more charged ones in fractions II and III. Polyacryl amide gel electrophoresis of fraction I shows a single major band of 39 kdaltons; in fractions II and III a major band of 42 kdaltons and fainter bands in the range 62–68 kdaltons are observed.

Fraction I has the highest specific binding for [³H]-acetylcholine (7.1 nmol/mg protein) and [³H]α-bungarotoxin (5.2 nmol/mg protein). The nicotinic nature of this proteolipid was demonstrated by blocking experiments. The Scatchard plot showed two affinity sites for [³H]-acetylcholine (Kd₁ = 3nM and Kd₂ = 1.1 μm). 4-(N-maleimido) pheny1 [³H]trimethylammonium specifically labeled the 39 kdaltons band.

The possible factors involved in the fractionation of proteolipids are discussed. The findings suggest that the 39 kdaltons polypeptide contains the receptor site for acetylcholine and that the other proteolipid components may play a different function in the membrane.     

Metabolism of Angiotensin Peptides by Neuronal and Glial Cultures from Rat Brain

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.     

Electrophoretic studies on the phosphorylase isozymes.

The electrophoretic method of Davis, Schliselfeld, Wolf, Leavitt and Krebs (1967) for phosphorylase isozymes has been modified. By this method, five isozymes were separated in various organs of rat and pig and were disignated as phosphorylase L, LI, I, II and III. The L and III enzymes were the only forms found in liver and skeletal muscle, respectively, while the I enzyme was dominant in brain, uterus, lung and small intestine, which also contained some fractions of the II and III enzymes. The I enzyme was also dominant in adrenal, ovary and kidney, but these organs contained the L+II or L+LI as minor components. The L and LI were richly found in spleen and leukocytes of adult rats and pigs and in liver of newborn rats. Such organ-specific heterogeneity of phosphorylase was confirmed by the immunological tests with the antibodies prepared against phosphorylases I, III and L. The II and LI enzymes were found to be the hybrid molecules between the I and III enzymes, and between the I and L enzymes which have been previously reported as unhybridizable, respectively. In view of the above findings, it was concluded that the rat and pig possessed at least five molecular forms of phosphorylase.     

Implication of persimmon fruit hemicellulose metabolism in the softening process. Importance of xyloglucan endotransglycosylase

Hemicellulosic polysaccharides from persimmon fruit ( Diospyros kaki L.) pericarp were extracted from depectinated cell walls with 0.5, 1 and 4 M KOH at different stages of development: (I) maximal growth corresponding to the first sigmoidal growth phase; (II) cessation of growth corresponding to the lag between the first and the second sigmoidal phases; (III) maximal growth corresponding to the second sigmoidal phase; and (IV) cessation of growth when the fruit had reached its maximum size and the change in colour (green to red) had taken place. During fruit development the amount of total hemicelluloses per unit dry mass cell wall decreased twofold. Xyloglucan was present in the three hemicellulosic fractions, and also decreased with fruit age, although its amount relative to other hemicelluloses increased. The amount of xyloglucan was especially high in the hemicelluloses extracted with 4 M KOH, representing more than 50% at stages III and IV. The average molecular mass of xyloglucan increased from stage I through stage II (0.5 M hemicellulosic fraction) or through stage III (I and 4 M hemicellulosic fractions) and decreased after that. The xyloglucan endotransglycosylase (XET: EC 2.4.1.-) activity was measured as the incorporation of [³H]XXXGol (reduced xyloglucan heptasaccharide labelled at position 1 of the glucitol moiety) into partially purified persimmon fruit xyloglucan. XET specific activity increased greatly between stages I and II. The importance of this enzyme during fruit ripening is discussed.     

Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp. nov

Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied. The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto. Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity. As a result, Cluster I was split into Groups I and II. Group I included the type strain of P. fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains. Group II contained two strains, and the level between the two strains ranged from 91 to 100%. Group III consisted of one strain. Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T). Clusters II and III corresponded to Groups III and IV, respectively. The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity. The strains of Groups I, II, and III had ubiquinone 9 as the major quinone. According to numerical analysis by the use of 133 phenotypic characteristics, the seven P. fulva strains were split into four phenons (Phenons I to IV). The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized. Consequently, Group I was regarded as P. fulva because the type strain (NRIC 0180(T)) of this species was included in this group. Strains in Group II were identified as a new species, Pseudomonas parafulva sp. nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501). NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp. nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246). NRIC 0182 in Group IV was identified as P. straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Beta-endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.