A teleost (Tilapia mossambica) gonadotropin that resembles luteinizing hormone.

A purified gonadotropin preparation was obtained from pituitaries of a teleost fish ($\text{Tilapia}$$\text{mossambica}$). This gonadotropin was found to resemble LH in that it behaved identically to mammalian and non-mammalian LHs in several chromatographic systems, and stimulated testerone production in isolated rat Leydig cells. In this assay, specific for LH, the $\text{Tilapia}$ gonadotropin was less potent than mammalian LHs but significantly more active than avian, reptilian or amphibian LHs. The $\text{Tilapia}$ gonadotropin was found to be a glycoprotein; preliminary amino acid composition data show resemblances to both mammalian and non-mammalian LHs.     

Isolation and characterization of luteinizing hormone from amphibian (Rana catesbeiana) pituitaries.

We report here the first isolation of an anterior pituitary hormone from an amphibian species, the bullfrog ($\text{Rana catesbeiana}$). Highly purified luteinizing hormone was isolated from alkaline extracts of bullfrog pituitaries by salt fractionation, chromatography on ion-exchangers and gel filtration. Characterization studies show the hormone to contain 9% carbohydrate and to possess an amino acid composition similar to ovine luteinizing hormone. Sedimentation-velocity experiments in the ultracentrifuge indicate that the bullfrog gonadotropin dissociates in acidic solution and is composed of subunits. Bullfrog luteinizing hormone is highly active in an $\text{in vitro}$ toad ovulation assay and also ellicits testosterone production $\text{in vitro}$ from isolated rat testis Leydig cells.     

Studies on the purification of locust adipokinetic hormone

$\text{Locusta}$ adipokinetic hormone (AKH) has been purified from glandular lobes of corpora cardiaca by a series of chromatographic steps that have allowed a 58 to 77% recovery of activity. Methanolic extracts of the glandular lobes were fractionated on Sephadex LH-20 and then on thin-layer plates. The average recovery of AKH activity after separation on Sephadex LH-20 approached 100%, whereas recoveries of between 58 to 77% were obtained after TLC on cellulose plates. In practice, the highest recoveries were obtained with separations of large amounts of the hormone on cellulose plates. In the course of this investigation it was found that AKH activity can be extracted efficiently from aqueous solutions using Florisil. The hormone activity was recovered from the Florisil by eluting with 80% methanol. The purified hormone was found to be homogeneous in composition when subjected to rechromatography on Sephadex LH-20 and to amino acid analysis of the acid hydrolysate. From the amino acid analysis we estimate that one pair of glandular lobes contains on average 188 pmoles of AKH.     

Cell-free synthesis of angler fish preproinsulin: Complete amino acid sequence of the signal peptide

Messenger ribonucleic acid isolated from angler fish ($\text{Lophius}\text{americanus}$) islets of Langerhans was translated in the wheat germ cell-free protein synthesizing system containing different combinations of radioactive amino acids. Preproinsulin (～ 11,000 daltons) was identified amongst the translation products, by sodium dodecyl sulfate gel electrophoresis, and subjected to microsequencing techniques. The fish preproinsulin was found to possess an NH₂-terminal signal peptide of 24 amino acids, with regions of homology to human, rat and chicken preproinsulin signal sequences.     

The linkage of teleost skin keratan sulfate to protein

The linkage of teleost skin keratan sulfate to protein was investigated. Afeter its exhaustive digestion with pronase, peptidokeratan sulfate was obtained with aspartic acid as the predominant amino acid. The N-terminal of the amino acid residues of the preparation was dansylated, and the carbohydrate-peptide linkage fragment was isolated, with the aid of fluorescence, by sequential digestion with Flavobacterium endo-β-galactosidase, β-galactosidase, β-N-acetylhexosaminidase and endo-β-N-acetylglucosaminidase D, followed by Bio-Gel p-4 column chromatography. The structure of the dansylated fragment thus obtained was identified dansylated asparaginyl $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$. Treatment of the dansylated keratan sulfate peptide with almond glycopeptidase, which specially cleaves thet asparaginyl $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$ linkage in the glycoproteins, also showed asparaginyl $\text{N-}\text{aceytyl-}\text{D}\text{-glucosamine}$ linkage to be in the core region of this keratan sulfate. We conclude that teleost skin keratan sulfate is bound to protein via an N-glycosyl linkage between $\text{N-}\text{acetyl-}\text{D}\text{-glucosamine}$ and asparagine. The keratan sulfate core apparently consist of trimannosyl-N,N′-diacetylchitobiose units, considering the specificity of endo-β-N-acetylglucosaminidase D.     

Transfer of methionyl residues by leucyl,phenylalanyl-tRNA-protein transferase

Leucyl, phenylalanyl-tRNA-protein transferase also catalyzes transfer of methionyl residues as indicated by (i) copurification over a 1000-fold range of transfer activities for all three amino acids and (ii) loss of methionyl transfer activity in a mutant of $\text{E.}\text{coli}$ lacking the transferase and reappearance of this activity in a transferase revertant. The purified enzyme was found to use Met-tRNA_m^Met in preference to Met-tRNA_f^Met as donor substrate. Peptides containing a basic amino acid at the NH₂-terminus functioned as acceptors for the transfer of methionyl residues.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Bombesian alters behavioral thermoregulation in fish

Bombesin was microinjected into the ventricular system of a teleost fish, $\text{Catostomus commersoni}$, in order to evaluate its effects on behavioral thermoregulation in a horizontal thermal gradient. General locomotor activity was also examined. Results demonstrated that bombesin decreased the behaviorally selected internal body temperatures by 5.0–7.0°C relative to controls and that the latency of the response was dose-dependent. The peptide did not change locomotor activity. Results are discussed in relation to body temperature setpoint(s) and possible implications for endotherm thermoregulation.     

Bacteriophage-induced lytic enzyme which hydrolyzes L-alanine-D-glutamic acid peptide bond in peptidoglycan

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.     

A difference in the in vitro accumulation and metabolism of testosterone -1,2-3H by the rat prostate gland following incubation with ovine or bovine prolactin

The $\text{in vitro}$ actions of ovine or bovine prolactin on the accumulation and metabolism of testosterone-1,2-³H by various lobes of the rat prostate gland were examined. Ovine prolactin at a concentration of 2 IU/ml of incubation media significantly increased the accumulation of total radiosteroid by the anterior and dorsal lobes of the rat prostate gland. Although the levels of both testosterone-1,2-³H and its principle metabolite 5α-dihydrotestosterone-1,2-³H were increased by this concentration of ovine prolactin, the ratio of the two steroids was not altered. Ovine prolactin did not alter either the accumulation or metabolism of testosterone-1,2-³H by the ventral or lateral lobes of the rat prostate gland.In contrast to ovine prolactin, bovine prolactin in a wide range of concentrations (0.02–8 IU/ml) significantly reduced the accumulation of total labeled radiosteroid by the dorsal lobe of the rat prostate gland. The reduction in total radiosteroid was reflected in concomitent decreases in both testosterone-1,2-³H and 5α-dihydrotestosterone-1, 2-³H. The anterior lobe responded with similar reductions but only at relatively high concentrations of bovine prolactin (4 and 8 IU/ml). No significant alterations were observed in either the ventral or lateral lobes as a result of incubation with bovine prolactin.     

Purification of penicillin-insensitive DD-endopeptidase, a new cell wall peptidoglycan-hydrolyzing enzyme in Escherichia coli, and its inhibition by deoxyribonucleic acids.

Activity of a penicillin-insensitive $\text{DD}$-endopeptidase that splits the $\text{D}$-alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of $\text{Escherichia coli}$. The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments.     

Insect yolk protein precursor, a juvenile hormone induced phosphoprotein

The juvenile hormone induced vitellogenic female-specific protein of $\text{Leucophaea}$$\text{maderae}$ was isolated from hemolymph of egg maturing females on DEAE or QAE anion-exchange columns. Minor contaminants could be removed by centrifugation to equilibrium on CsCl gradients. The buoyant density of this purified protein is 1.344 g/ml. It is a lipophosphoprotein of low phosphorus (0.14%) content. Essentially all ³²P label from in vivo labelled protein was recovered in phosphoserine. The amino acid residues of the vitellogenic protein compare well with the purified yolk protein.     

Studies on prolactin. Selective reduction of the disulfide bonds of the ovine hormone.

Methods for selective reduction of the disulfide bonds in ovine prolactin are reported. Cleavage of all three disulfide bonds abolishes biological activity and denatures the hormone. Reduction-carbamidomethylation of one or two of the disulfide bridges does not diminish the biological activities in the pigeon crop-sac and mouse mammary gland bioassays. When compared to the native hormone, monomers of these two partially reduced-carbamidomethylated derivatives also show only modest changes in properties measured by exclusion chromatography, circular dichroism, and immunological cross-reactivities. However, cleavage of cystine-4--11 and cystine-191--199, followed by carbamidomethylation, destroys the biological activity of this derivative in a teleost fish bioassay (Gillichthys urinary bladder). In contrast, reduction of cystine-4--11 actually increased the teleost potency of this derivative compared to the intact hormone. Since teleost prolactin appears to lack a homologue to the cystine-4--11 disulfide bond in the amino-terminal loop of the ovine hormone, selective reduction of this bond in ovine prolactin may produce a derivative whose properties more closely resemble the fish hormone.     

An unusual histone H4 gene from Tilapia nilotica exhibiting characteristics of both a replication-dependent histone and a basal-expression histone: evolutionary considerations

The nucleotide sequence of a histone H4 gene from the fish Tilapia nilotica was determined. The deduced amino acid sequence is identical to that of H4 from the trout, Salmo gairdnerii. The 3′ untranslated region of the Tilapia gene exhibits a unique combination of structural features each of which is usually associated with either a replication-dependent histone or a basal-expression histone, but not with both. The direction of nucleotide substitutions in the Tilapia and Salmo lineages was inferred. The Tilapia gene was found to evolve faster and to exhibit a more biased pattern of substitution and codon usage than its Salmo homologue. This combination of features cannot be explained by either mutation or purifying selection. The rapid embryonic development of Tilapia prompts us to suggest that the molecular evolution of the histone H4 gene is driven by fixation of advantageous synonymous mutations.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Characterization of NADPH-cytochrome P-450 reductase from house flies (Musca domestica,L.) susceptible and resistant to insecticides,and the blow fly (Phormia regina,meigen)

NADPH-cytochrome $\text{c}$ reductase was purified by affinity chromatographic techniques from microsomes prepared from the abdomens of insecticide-resistant (R) and -susceptible (S) house flies Musca domestica and of the black blow fly Phormia regina. Data are presented which describe (1) the ability of the purified enzymes to support an in vitro reconstitution of mono-oxygenase activity, (2) the changes in activity of these preparations observed in buffers of varying ionic strength, (3) comparative kinetic behaviour between microsomal and purified forms of the enzymes, (4) the immunochemical characteristics of these preparations, and (5) their amino acid composition. The reductases from the three sources were found to be very similar in all of these tests. Substrate binding constants were 5 μM for NADPH, 12 μM for cytochrome $\text{c}$, and the catalytic mechanism was interpreted as ordered Bi Bi. The inhibitory constant of the reductase from the resistant fly for 2′-AMP, an analogue of NADP⁺, was 187 μM; whereas no assciation of the inhibitor was observed below concentrations of 400 μM for the enzyme of either the susceptible house fly or the blow fly. However, the data are insufficient to suggest that the reductase is a significant factor in insecticide resistance. Compared to the same enzymes from rat and rabbit liver, the insect reductases show a different ionic strength optimum (0.14), have distinct antigenic determinants, and have different levels of acidic and basic amino acids in the membrane-binding peptide.