Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse

Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms were found to contain an equivalent amount of hexose (9.0-9.2%). Isohormones eLH-B and eLH-C, however, possess more sialic acid than eLH-A (6.6-6.7%, vs. 4.5%). The eLH-A and eLH-B preparations contain a similar amount of hexosamine, which is slightly lower than the amount of eLH-C (8.8-9.1% vs. 11.2%). No differences were noted between the isohormones by rat Leydig cell LH bioassay, equine testis LH radioreceptor assay (RRA) or calf testis follicle-stimulating hormone (FSH) RRA. Slight, but nonsignificant, variations were noted between preparations in an eLH radioimmunoassay (RIA). Although chemical variations were detected between the eLH isoforms, no significant differences were observed in in vitro biological and immunological activities. The differences detected in sialic acid content raises the possibility that differences in in vivo clearance rates may exist.     

Purification of lutropin and follitropin in high yield from horse pituitary glands

A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitaries, respectively. Subunits were prepared by dissociation in 8 M guanidine HCl followed by either gel filtration (eLH) or gel filtration followed by QAE-Sephadex chromatography (eFSH). The hormones and their subunits were characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, NH2-terminal analysis, and by LH and FSH radioligand receptor assays.     

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.     

猪生长激素的提纯及其生物学活性和某些理化性质的研究

通过调pH抽提、硫酸铵分级分离和DEAE-纤维素柱层析等方法从猪垂体中提取了生长激素。平均产率约为0.5g生长激素/1000g鲜垂体。用去垂体大白鼠胫骨测定法测得共促生长效应为1.84。提取的生长激素在高pH不连续缓冲体系聚丙烯酰胺凝胶电泳时呈现两条带;Sephadex G75柱层析得到一个峰;SDS聚丙烯酰胺凝胶电泳呈现一条带,其分子量约为21,900道尔顿;聚丙烯酰胺凝胶等电聚焦电泳呈现五条带,主带在pH7.8处;DNS法测定其N-末端氨基酸为苯丙氨酸一种。此外,还测定了它的氨基酸组成。     

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

Summary A method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.     

Human luteinizing hormone. Isolation and characterization of the native hormone and its alpha and beta subunits.

A new procedure is described for the isolation of the alpha and beta chains of the hormone. In this method, thenative hormone is incubated in acidic urea and the chains are then separated by ion-exchange chromatography. The amino-terminal residue of the alpha subunit is valine. The carboxy-terminal end of the alpha subunit is of variable length. No amino-terminal residue was detected for the beta chain; glycine was found at its carboxy-terminal end by the selective titration method. The amino acid and carbohydrate compositions of the hormone and both subunits are presented. The beta chain contains sialic acid and is devoid of galactosamine in contrast to the beta subunits of other species. Contamination of our human lutenizing hormone preparation by other pituitary glycoprotein hormones such as thyroid-stimulating hormone and follicle-stimulating hormone amounted to 0.5 and 0.25 percent by weight respectively. Cross-contamination of the initial alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 4.1 and 2 percent by weight respecitively. Further extensive purification of these subunit preparations was then performed by means of affinity chromatography using immunosorbants. The final preparations exhibited a residual cross-contamination amounting to 0.2 and 0.02 percent by weight for the alpha and beta subunits respectively.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Amino acid sequence of corticotropins from seiwhale (Balaenoptera borealis) and pinwhale (Balaenoptera physalus)]

The corticotropins from two species of whales, e. g. seiwhale (Balaenoptera borealis) and finwhale (Balaenoptera physalus) were subjected to hydrolysis by trypsin, chymotrypsin and pepsin. The peptide fragments were separated by gel-filtration through Sephadex and partition paper chromatography. The study of the amino acid sequence of the peptides obtained allowed to establish the primary structure of corticotropin from both species, which was found structurally identical to human corticotropin.     

Isolation and characterization of beta-lipotropin and adrenocorticotropin from turkey pituitary glands

Beta-Lipotropin and corticotropin have been isolated in highly purified form from turkey pituitary glands. The isolated hormones were characterized by NH2-terminal residue, amino acid and sequence analyses. Their hormonal activity and immunoactivity were also investigated.     

Some Researches on Histones

The histone extracted from calf thymus glands is a complex system of proteins, which can be fractionated by chromatography on carboxymethyl cellulose columns into three principal fractions (1) very lysine-rich, (2) moderately lysine-rich, (3) arginine-rich. When examined by starch gel chromatography each of these gives more than one band. Methods have been devised for further separation of the components in some cases. The components show characteristic differences in end groups and certain amino acids as well as in their basic character. Histones extracted from various rat tissues can be separated into similar fractions, of which the amino acid analyses are similar to those derived from calf thymus, within the experimental error. To this extent, no species or tissue specificity of the fractionated histones was observed. Although all the histone fractions contain approximately one basic amino acid to three non-basic amino acids their structure is not regular, as Phillips has shown that in certain fractions the number of non-basic groups between two basic groups may vary from 0 to seven or more. The possible functions of histones are discussed.     

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.     

Isolation of corticotropin-beta-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-beta-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824-2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel electrophoresis, using immunoprecipitated and electrophoretically purified [125I]corticotropin-beta-lipotropin common precursor as a marker. The preparation was judged homogeneous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and beta-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-beta-lipotropin common precursor possessed specific activities of 116 micrograms of immunoreactive corticotropin and 210 micrograms of immunoreactive beta-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Changes in elastin composition in aorta of spontaneously hypertensive rats (SHR)

Tropoelastin and elastin preparations obtained from aortae of spontaneously hypertensive rats (SHR) show an increased proportion of polar amino acids (aspartic acid, glutamic acid, arginine and tyrosine). The content of these amino acids is 1.43-3.04 times higher in SHR rats than in similar elastin or tropoelastin preparations obtained from normotensive animals. On the other hand elastin and tropoelastin preparations obtained from SHR rats show a lower frequency of the Val-Pro sequence; this was found to be 35.93 per 1000 amino acid residues in SHR rats as compared to 51.04 per 1000 amino acids in the preparations obtained from control animals. Since similar differences were found not only in elastin preparations but also in tropoelastin, contamination of these preparations with an acidic protein seems unlikely. In general the results obtained are similar to those seen in animals kept on a long term high fat diet. It appears feasible to suggest that these differences are caused by a changed proportion of two different elastin type.     

The first identified neuropeptide in the insect order Megaloptera: a novel member of the adipokinetic hormone family in the alderfly Sialis lutaria

This is the first report on the structural identity of a neuropeptide of the insect order Megaloptera. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca glands of the alderfly, Sialis lutaria. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Thr-Phe-Thr-Pro-Ser-Trp amide. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by comparing retention time on reversed-phase HPLC and establishing co-elution with the synthetic Leu(2)-form which also had exactly the same MS(2) mass spectra as the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which has already more than 40 members. Interestingly, the primary structure is identical to that predicted from genome information for the adipokinetic hormone of the yellow fever mosquito, Aedes aegypti. Since alderflies are not known for their active flight metabolism but produce a rather high number of eggs, it is anticipated that the alderfly is a good study object to establish a possible role of the novel peptide to regulate fat mobilization from the fat body and transport into the egg, thereby playing a role in the control of reproductive processes.     

Circular dichroism of corticotropin, fragment 1-24, and model compounds. An assessment of the contributions of the peptide chromophore and armoatic residues.

The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1–24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.     

Chemical structure and properties of duck and goose fibrinogen

Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented.     

Purification and Characterization of High Molecular Weight Human Pituitary Prolactin

A large form of human prolactin (molecular weight 150 000–170 000) was purified from the residue remaining after extraction at neutral pH of homogenized frozen pituitaries. This purification involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, and hydrophobic interaction chromatography on pentyl-Sepharose 4B. The procedure was followed by radioimmunoassay. The large form of prolactin was prepared both from fresh and from long-term stored residues. In the latter case the final yield was considerably higher. By zone electrophoresis in agarose suspension the prolactin preparation was separated into four or five immunoactive components. In sedimentation equilibrium analysis in the ultracentrifuge, however, these isohormones showed heterogeneity, which was suggested to be caused by dissociation. Evaluation of data obtained from the bottom region of the cells gave molecular weight values of the components in the range of 160 000 – 180 000. One of the is hormones s further studied and exhibited bioactivity in the local crop-sac assay and showed an amino acid composition closely similar to that of the native monomer prolactin. The high molecular weight prolactin was partially dissociated by treatment with 50% ethylene glycol or 1 M propionic acid or 6 M guanidine hydrochloride. Molecular sieve chromatography in the presence of these dissociating agents, resolved the prolactin activity into three separate peaks. The most retarded fraction, which eluted in a position corresponding to that of native monomer prolactin was characterized by electrophoresis and amino acid analysis. The results were supporting evidence that the dissociation procedure gave a monomer which had a lower amide content than the native monomer. Furthermore, its specific immunoactivity was 2–3 times higher than the activity of the intact large form.     

Molecular cloning of crustacean pigment dispersing hormone precursor.

The cDNA encoding the precursor of the pigment dispersing hormone (PDH) of the shore crab, Carcinus maenas, was isolated and sequenced. The precursor consists of a putative 22 amino acid signal peptide, a putative 33 residue peptide of unknown function, and the 18 amino acid mature PDH, followed by a Gly residue which serves as a possible amide donor. The deduced mature PDH amino acid sequence is identical to those of Uca pugilator and Cancer magister, previously determined by Edman degradation.     

Phylogeny of neurohypophyseal hormones in birds: microidentification of mesotocin and vasotocin in the ostrich (Struthio camelus)

Ostrich (Struthio camelus) neurohypophysial hormones have been isolated from 5 freeze-dried posterior pituitary glands. Purification has involved three steps: a first molecular sieving on Sephadex G-75 for eliminating proteins, a second molecular sieving on Bio-Gel P4 for separating the two active principles and a high pressure reverse-phase liquid chromatography (HPLC) on Nova-Pak C 18 with an 10 mM acetate-acetonitrile gradient for isolating each hormone. The active peptides have been identified by their retention time in HPLC and their amino acid composition. Mesotocin and vasotocin have thus been characterized. Although the phylogeny of Ratites is disputed, in particular their possible common origin with Carinates, which include most of the living birds, species of the first sub-class seem to have the same neurohypophysial hormones as those of the second.