Hugh D. Niall

Metabolism of parathyroid hormone: Physiologic and clinical significance

Abstract Recent findings concerning the metabolism of parathyroid hormone (PTH) and the heterogeneity, or multiple forms, of circulating PTH have spurred widespread investigation of the nature, origin and physiologic significance of the circulating fragments of PTH. Studies of endogenous PTH in man and cow, and of bovine PTH administered to dogs, have been performed utilizing sensitive, specific immunochemical and radiochemical technics. These studies indicate that fragments of the hormone are responsible for most of the immunoreactive PTH (iPTH) detected in the general circulation and that hormonal cleavage occurs after secretion but not in the intravascular space. Studies of parathyroid effluent plasma have established that the principal secretory product of the parathyroid glands is intact hormone; whether other immunoreactive forms of the hormone are also secreted from the gland is uncertain. In all species studied, the predominant form of circulating hormone is the large fragment consisting of the middle and COOH-terminal portions of the molecule; it lacks more than a third of the NH 2 -terminal portion. Because the NH 2 -terminal portion of the hormone sequence is required for biologic activity, this large fragment must be biologically inactive. Analysis of cleavage patterns of intact hormone indicates that an NH 2 -terminal fragment containing the necessary structural requirements for biologic activity may be produced by this cleavage. This may be of considerable physiologic significance regardless of whether this NH 2 -terminal fragment circulates or is present only outside the intravascular space. Although the significance of hormone metabolism is still unclear, the present findings are helpful in the application and interpretation of radioimmunoassays for PTH.

A method for preparing β-hCG COOH peptide-carrier conjugates of predictable composition

Abstract A method for coupling peptides related to the beta subunit of human chorionic gonadotropin (hCG) to macromolecular carriers that permits covalent conjugation in a predictable fashion was developed. Peptides representing hCG beta subunit residues 109–145 and 111–145 were coupled to tetanus toxoid, flagellin, synthetic polypeptides and Ficoll. Carrier compounds containing amino groups but devoid of sulfhydryl groups were reacted with 6-maleimido caproic acyi N -hydroxy succinimide ester (MCS) under conditions that result in the bifunctional reagent attached to carrier amino groups with the stable maleimido group free. Subsequently, peptides containing sulfhydryl groups were reacted with the reagent modified carrier whereby the peptides coupled to the carrier via the reaction between sulfhydryl groups on peptide and the maleimido group on carriers. Peptides devoid of sulfhydryl groups were thiolated using homocysteine thiolactone. The efficiency of coupling was confirmed by amino acid analysis and SDS polyacrylamide electrophoresis. Results indicated that the coupling of peptides to carriers could be regulated by the number of moles of MCS reacted with carrier to yield free maleimido groups. Sulfhydryl group reaction with maleimido groups was stoichiometric. Conjugates prepared by this method were used to immunize rabbits and significant levels of antibodies to the hCG peptides have been attained.

The Structure and Biosynthesis of Epidermal Growth Factor Precursor

SUMMARY The structure of mouse submaxillary gland epidermal growth factor (EGF) precursor has been deduced from complementary DNAs. The mRNA is approximately 4800 bases and predicts prepro EGF to be a protein of 1217 amino acid residues (133×10 Mr). EGF (53 amino acid residues) is flanked by polypeptides of 188 and 976 residues at its carboxy and amino termini, respectively. The amino terminus of the precursor contains seven cysteine-rich peptides that resemble EGF. Towards the carboxy terminus is a 20-residue hydrophobic membrane spanning domain. The mid portion of the EGF precursor shares a 33 % homology with the low density lipoprotein receptor, which extends over 400 amino acid residues. These features suggest that EGF precursor could function as a membrane-bound receptor. RNA dot-blot analysis and in situ hybridization show EGF mRNA to be abundant in the submaxillary gland, kidney and incisor tooth buds. Lower EGF mRNA levels were found in the lactating breast, pancreas, small intestine, ovary, spleen, lung, pituitary and liver. In the kidney EGF mRNA was most abundant in the distal convoluted tubules. Analysis of EGF precursor biosynthesis in organ culture of the submaxillary gland and kidney showed differential processing of the precursor in the two tissues. In the submaxillary gland immunoreactive low molecular weight EGF was produced, but in the kidney the high molecular weight precursor was not processed. In the distal convoluted tubule of the kidney EGF precursor may act as a receptor that is involved in ion transport.

The nucleotide sequences of baboon chorionic gonadotropin β-subunit genes have diverged from the human

The placental glycopeptide hormone chorionic gonadotropin (CG) is involved in establishing and maintaining pregnancy. The hormone consists of two different non-covalently associated subunits termed alpha and beta. In man there are seven closely linked genes coding for beta CG-like peptides, but only three of these appear capable of expression in the placenta. The organization of beta CG-like genes in man and baboon appears to be similar. We demonstrate here that the baboon genome contains multiple copies (at least five) of beta CG-related genes, and that these genes are closely linked in the genome. Nucleotide sequence data from several beta CG cDNA clones indicates that at least two of these beta CG-related genes are expressed in the baboon placenta. Analysis of beta CG sequences from baboons and human subjects demonstrates that these genes have diverged markedly between species. In contrast, these sequences are remarkably homogeneous within their respective genomes. Gene conversion events may be responsible for retaining the high degree of identity among the various beta CG gene family members. Knowledge of beta CG sequences from baboon may lead to development of a long-term antipregnancy vaccine. The ability of CG antibodies to interfere with the maintenance of pregnancy can now be investigated within a homologous system.

Characteristics of antibodies raised to carboxy-terminal peptides of hCG beta subunit

Abstract Natural and synthetic peptides representing COOH terminal sequences of the beta subunit of human chorionic gonadotropin (hCG) were coupled to tetanus toxoid and rabbits immunized with the conjugates. Sera were evaluated for antibody levels, antibody affinity and antibody class. Conditions for appropriate estimation of these parameters were also studied. Findings revealed that iodination of hCG or peptides did not alter their immunological properties and that reaction of labelled antigens with antisera require differing periods of time for the establishment of equilibrium. Peak titers to the hCG antigens were reached at 70 days of immunization. Antibodies to natural peptide 109–145 of β-hCG and synthetic peptides 109–145, 111–145, and 115–145 reacted to hCG approximately 90% as well as they reacted to the peptide used for immunizations whereas antibodies to synthetic peptide 125–145 reacted only 60–70% as well. Mean antibody affinities to peptides were not significantly higher than affinities of the same sera to hCG. The affinity of antisera to synthetic peptide 125–145 to both antigens and the mean ratio of hCG affinity : peptide affinity was somewhat lower than those to longer peptides. After 70 days of immunization, antibodies were predominantly of the IgG class. The findings indicate that antibodies raised to natural or synthetic peptides of the COOH region of hCG beta subunit are highly cross-reactive to native hCG but have affinities somewhat lower than those generated to the intact hormone or its beta subunit.

Insulin-related growth factors

A distinction can be drawn between primary hormones (messengers) (rapidly acting and using cyclic nucleotides as second messengers) and secondary hormones, which generally act over a longer time period and characteristically provide trophic stimulation to their target cells. Insulin and three related polypeptide growth factors (relaxin, nerve growth factor and insulin-like growth factor) fall into the second category. They share features of primary and three-dimensional structure as well as certain functional similarities and in all probability evolved from a common ancestral gene.

STUDIES ON THE PURIFICATION OF OVINE INHIBIN

There is now good evidence that a nonsteroidal substance, inhibin, is produced by the gonads to regulate the secretion of follicle stimulating hormone (FSH) by the pituitary At present there is little agreement about the chemical characteristics of inhibin. Some claim it is a basic protein with a molecular weight of about 20,000 and is easily isolated from extracts of testes, seminal plasma, or follicular fluid by conventional purification t echn iq~es .~ -~ Others find it is larger and difficult to p ~ r i f y . ~ l ~ This communication reviews our recent studies on the nature of inhibin from ovine rete testis fluid (RTF) and attempts at purification.

Evidence for Proteolysis during Purification of Relaxin from Pregnant Sow Ovaries

The proteolytic degradation of relaxin during its isolation from pregnant sow ovaries has been examined. Ovaries from pregnant sows were selected and divided into three groups according to the stages of pregnancy. Each group was extracted with and without protease inhibitors. It was found that protease(s) were present in all groups of ovaries and that a 2-4 fold increase in yield of total relaxin was obtained when isolation and purification was carried out in the presence of protease inhibitors. However the ratio of the three forms of relaxin remain unchanged.

Renin gene expression in vessels of the ovine renal cortex.

Using hybridization histochemistry, a technique which localizes specific mRNA populations in tissue sections with a 700 base pair recombinant DNA probe which codes for ovine renin, we have localized renin gene expression in the afferent arteriole of the juxtaglomerular apparatus (JGA) in the sheep renal cortex. Specific labelling representing renin gene expression was also found at a distance from the glomerular tuft in the walls of the afferent arteriole and also in cells in the medial layer of larger vessels of the renal cortex, specifically the interlobular arteries. These observations provide morphological evidence of renin gene expression at these sites and, combined with ultrastructural and immunocytochemical evidence suggest that renin is synthesized and stored in the afferent arteriole either within the JGA or at a distance from the glomerulus, and in the smooth muscle coat of the interlobular arteries in the sheep kidney.

Rat relaxin: insulin-like fold predicts a likely receptor binding region

Abstract The amino acid sequences for the ovarian hormone relaxin, now determined for pig, rat and shark, indicate that the molecule may have an internal structure similar to that of insulin. The combined results from six secondary structure prediction methods applied to the sequences of both relaxin and insulin support the concept of a similar folding for the B chain between the disulphide bridges. Model building with a computer graphics system has shown that the rat relaxin sequence cannot be superimposed on the 2Zn insulin structure without close contacts occurring between the residues in the central core. However, the residues can be accommodated in the more open framework assumed by 4Zn insulin (molecule I). With the relaxin models built according to the insulin fold, surface residues shared by the three relaxin sequences (B9(Arg), B13(Arg), A13 and A14 (Lys or Arg)) all lie in a localized area on the molecule. This group of residues focuses attention on a larger area on the molecules surface which may well be the receptor binding site.