DEVELOPMENT OF HOMOLOGOUS IMMUNOLOGICAL ASSAYS FOR HUMAN PARATHYROID HORMONE

1980 ◽  
Vol 85 (1) ◽  
pp. 161-170 ◽  
Author(s):  
R. M. MANNING ◽  
G. N. HENDY ◽  
S. E. PAPAPOULOS ◽  
J. L. H. O'RIORDAN
Keyword(s):  
Parathyroid Hormone ◽  
Trichloroacetic Acid ◽  
Terminal Region ◽  
Human Parathyroid Hormone ◽  
Antibody Assay ◽  
Amino Terminal ◽  
High Affinity ◽  
Carboxy Terminal Region ◽  
Immunological Assays ◽  
Carboxy Terminal

SUMMARY Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1–34 peptide (PTH-(1–34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule. Highly purified human PTH-(1–84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1–34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.

scholarly journals THE DISTRIBUTION OF ANTIGENIC DETERMINANTS IN RAT SKIN COLLAGEN

1971 ◽  
Vol 133 (6) ◽  
pp. 1309-1324 ◽  
Author(s):  
Herbert Lindsley ◽  
Mart Mannik ◽  
Paul Bornstein
Keyword(s):  
Terminal Region ◽  
Antigenic Determinants ◽  
Collagen Molecule ◽  
Rat Skin ◽  
Amino Terminal ◽  
Skin Collagen ◽  
Native Collagen ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hyperimmune Rabbit

Immunological studies of rat skin collagen were carried out with a sensitive and quantitative radioimmunoassay. Hyperimmune rabbit antisera to rat skin collagen and isolated α2 chains were used. Iodine-labeled α chains and CNBr-produced peptides served as test antigens, and native collagen, α chains, and CNBr peptides were employed as inhibitors in the assay. The α1 and α2 chains were immunologically distinct. Although the α1 chain was not immunogenic, antibodies to α1 were detected in antisera to the intact collagen molecule. The major antigenic determinant of the α1 chain was located in α1-CB6 which constitutes the carboxy-terminal region of the chain. The α2 chain contained two non-cross-reacting antigenic determinants, one in the amino-terminal region (α2-CB1) and the other in the carboxy-terminal region (α2-CB5) of the chain. The native collagen molecule was less effective than isolated α chains in inhibiting binding of labeled peptides to antisera, indicating that antigenic determinants were less accessible in the triple helical molecule. These immunologic studies are consistent with preliminary comparative biochemical data which indicate that interspecies structural differences in collagen predominate at both the amino- and carboxy-terminal ends of the chains.

Human Parathyroid Hormone: Immunological Properties of the Amino Terminus and of Synthetic Fragments

1974 ◽  
Vol 47 (6) ◽  
pp. 567-576 ◽  
Author(s):  
G. N. Hendy ◽  
P. M. Barling ◽  
J. L. H. O'Riordan
Keyword(s):  
Parathyroid Hormone ◽  
Synthetic Peptides ◽  
Terminal Region ◽  
Amino Terminus ◽  
Human Parathyroid Hormone ◽  
Immunological Properties ◽  
Immunological Assay ◽  
Amino Terminal

1. Immunological assay systems specific to the amino-terminal region of parathyroid hormone were used to study the properties of human parathyroid hormone (HPTH 1-84) and of synthetic peptides. One of these peptides (HPTH 1-34A) had the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts (1974). The others (HPTH 1-34B and C, synthesized by different methods) had the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud (1972). 2. HPTH 1-84 and HPTH 1-34A behaved identically in a number of these systems. In some systems, especially those using antisera obtained by immunizing with HPTH 1-34A, differences were, however, found. These may be due to conformational or other physical differences or to the presence of an immunologically incomplete site in the fragment. 3. In systems in which HPTH 1–84 and HPTH 1-34A behaved identically the peptides with the sequence proposed by Brewer et al. (1972) were of low reactivity by 100–2000-fold.

scholarly journals Structure and Function Analysis of LIN-14, a Temporal Regulator of Postembryonic Developmental Events in Caenorhabditis elegans

2000 ◽  
Vol 20 (6) ◽  
pp. 2285-2295 ◽  
Author(s):  
Yang Hong ◽  
Rosalind C. Lee ◽  
Victor Ambros
Keyword(s):  
Caenorhabditis Elegans ◽  
Function Analysis ◽  
Cell Types ◽  
Terminal Region ◽  
Gene Products ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Terminal Domains

ABSTRACT During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternativelin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternativelin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.

Regulation of fibrillin carboxy-terminal furin processing by N-glycosylation, and association of amino- and carboxy-terminal sequences

1999 ◽  
Vol 112 (22) ◽  
pp. 4163-4171
Author(s):  
J.L. Ashworth ◽  
V. Kelly ◽  
M.J. Rock ◽  
C.A. Shuttleworth ◽  
C.M. Kielty
Keyword(s):  
Extracellular Space ◽  
Molecular Mechanisms ◽  
Terminal Region ◽  
Amino Terminus ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Fibrillin 1 ◽  
Carboxy Terminal Region ◽  
Efficient Processing ◽  
Carboxy Terminal

The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12–15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.

scholarly journals The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy.

1996 ◽  
Vol 134 (6) ◽  
pp. 1441-1453 ◽  
Author(s):  
W M Obermann ◽  
M Gautel ◽  
F Steiner ◽  
P F van der Ven ◽  
K Weber ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Terminal Region ◽  
M Protein ◽  
Amino Terminal ◽  
Thick Filaments ◽  
Carboxy Terminal Region ◽  
Oriented Parallel ◽  
Carboxy Terminal

The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.

scholarly journals The Carboxy-Terminal Region of apoA-I Is Required for the ABCA1-Dependent Formation of α-HDL But Not Preβ-HDL Particles in Vivo†

Biochemistry ◽  
2007 ◽  
Vol 46 (19) ◽  
pp. 5697-5708 ◽  
Author(s):  
Angeliki Chroni ◽  
Georgios Koukos ◽  
Adelina Duka ◽  
Vassilis I. Zannis
Keyword(s):  
Terminal Region ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Parathyroid hormone metabolism and its potential as a uremic toxin

1980 ◽  
Vol 239 (1) ◽  
pp. F1-F12 ◽  
Author(s):  
E. Slatopolsky ◽  
K. Martin ◽  
K. Hruska
Keyword(s):  
Renal Failure ◽  
Parathyroid Hormone ◽  
Chronic Renal Failure ◽  
Biological Effects ◽  
Uremic Toxin ◽  
Terminal Portion ◽  
Single Chain ◽  
Biochemical Alterations ◽  
Amino Terminal ◽  
Carboxy Terminal

Secondary hyperparathyroidism is a universal complication of chronic renal failure. It has been proposed that the markedly elevated levels of immunoreactive parathyroid hormone (i-PTH) in uremia may represent a “uremic toxin” responsible for many of the abnormalities of the uremic state. Plasma i-PTH consists of a mixture of intact hormone, a single-chain polypeptide of 84 amino acids, and smaller molecular weight hormonal fragments from both the carboxy- and amino-terminal portion of the PTH molecule. The hormonal fragments arise from metabolism of intact PTH by peripheral organs as well as from secretion of fragments from the parathyroid glands. The structural requirements for the known biological actions of PTH reside in the amino-terminal portion of the PTH molecule. Carboxy-terminal fragments, biologically inactive at least in terms of adenylate cyclase activation, hypercalcemia, or phosphaturia, depend on the kidney for their removal from plasma, and thus accumulate in the circulation in chronic renal failure. It is unknown at the present time if other biological effects of these carboxy-terminal fragments may contribute to some of the biochemical alterations observed in uremia. The most significant consequence of increased PTH levels in uremia is the development of bone disease characterized by osteitis fibrosa. In addition, it would appear that PTH plays an important role in some of the abnormal electroencephalographic patterns observed in uremia. This may be due to a potential role of PTH in increasing calcium content of brain. Parathyroid hormone also has been implicated as a pathogenetic factor in many other alterations present in uremia, i.e., peripheral neuropathy, carbohydrate intolerance, hyperlipidemia, and other alterations. Unfortunately, outstanding clinical research is lacking in this field and conclusive experimental data are practically nonexistent. Further studies are necessary if one is to accept the concept of PTH being a significant “uremic toxin.”

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