Large Molecular Forms Research Articles

Identification of Kinase-Phosphatase Signaling Modules Composed of p70 S6 Kinase-Protein Phosphatase 2A (PP2A) and p21-activated Kinase-PP2A

A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A's important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258-1261) and casein kinase IIalpha (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.

Alterations in the hypothalamic pituitary adrenal axis during pregnancy and the placental clock that determines the length of parturition

Although corticotrophin releasing hormone (CRH) was initially identified as a hypothalamic peptide it is also synthesised in the placenta and secreted into both the maternal and fetal circulation. The presence of large molecular weight forms in the placenta suggest that secretion may be constitutive rather that regulated. Placental CRH is bioactive but causes only modest increases in ACTH and cortisol in the pregnant woman because of agonist induced desensitisation of pituitary CRH receptors. CRH concentrations increase exponentially in maternal plasma as gestation advances. Elevated concentrations, compared with gestational age matched controls, occur in patients in preterm labour. The exponential curve describing the CRH increase is shifted to the left in women who will subsequently deliver preterm and to the right in women who will deliver post dates indicating that CRH is linked to a placental clock which determines the length of gestation. Maternal plasma CRH concentrations may be useful in identifying women at high risk of preterm delivery and CRH antagonists may prevent preterm labour. As significant CRH production by the placenta is restricted to primates future research must take into account the species specificity of the mechanisms regulating parturition.

The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase

C-CAM, a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family, appears as two co-expressed isoforms, C-CAM-L and C-CAM-S, with different cytoplasmic domains, that can form homo-dimers in epithelial cells. In addition, C-CAM-L has been found in large molecular weight forms suggesting posttranslational, covalent modification. Here we have investigated the possibility that the cytoplasmic domain of C-CAM-L can act as a transglutaminase substrate. Glutathione S-transferase fusion proteins of the cytoplasmic domains of rat and mouse C-CAM-L as well as free cytoplasmic domains, released by thrombin cleavage from the fusion proteins, were converted into covalent dimers by tissue transglutaminase. These results demonstrate that the cytoplasmic domains of rat and mouse C-CAM-L are substrates for tissue transglutaminase, and lend support to the notion that higher molecular weight forms of C-CAM-L are formed by transglutaminase modification.

Anti-Prolactin (PRL) Autoantibodies Cause Asymptomatic Hyperprolactinemia; Bioassay and Clearance Studies of PRL-Immunoglobulin G Complex

The causes of hyperprolactinemia are varied, but some cases are classified as idiopathic because of unknown causes. We examined whether anti-prolactin (PRL) autoantibodies can cause hyperprolactinemia, especially the asymptomatic type. Serum PRL in four women with anti-PRL autoantibodies and five control patients with prolactinoma was characterized by a sensitive enzyme immunoassay, Nb2-bioassay, gel chromatography, affinity chromatography for immunoglobulin G (IgG), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, and clearance studies using anesthetized rats. In four women with anti-PRL autoantibodies, serum immunoreactive PRL concentrations were elevated (326 +/- 216 micrograms/L, normal < 30 micrograms/L), and PRL (84 +/- 5.5%) mostly consisted of the large molecular form in which a significant amount of 23 kDa PRL (60.6 +/- 14.7%) was noncovalently bound to IgG. Although three of the four women lacked clinical symptoms of hyperprolactinemia such as amenorrhea and galactorrhea, the IgG-bound PRL was fully bioactive in vitro. It was cleared more slowly from circulation than free PRL. The data suggest that PRL forms a complex with IgG, and this probably results in delayed clearance of PRL and leads to hyperprolactinemia in women with anti-PRL autoantibodies.

Anti-prolactin (PRL) autoantibodies cause asymptomatic hyperprolactinemia: bioassay and clearance studies of PRL-immunoglobulin G complex.

The causes of hyperprolactinemia are varied, but some cases are classified as "idiopathic" because of unknown causes. We examined whether anti-prolactin (PRL) autoantibodies can cause hyperprolactinemia, especially the asymptomatic type. Serum PRL in four women with anti-PRL autoantibodies and five control patients with prolactinoma was characterized by a sensitive enzyme immunoassay, Nb2-bioassay, gel chromatography, affinity chromatography for immunoglobulin G (IgG), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, and clearance studies using anesthetized rats. In four women with anti-PRL autoantibodies, serum immunoreactive PRL concentrations were elevated (326 +/- 216 micrograms/L, normal < 30 micrograms/L), and PRL (84 +/- 5.5%) mostly consisted of the large molecular form in which a significant amount of 23 kDa PRL (60.6 +/- 14.7%) was noncovalently bound to IgG. Although three of the four women lacked clinical symptoms of hyperprolactinemia such as amenorrhea and galactorrhea, the IgG-bound PRL was fully bioactive in vitro. It was cleared more slowly from circulation than free PRL. The data suggest that PRL forms a complex with IgG, and this probably results in delayed clearance of PRL and leads to hyperprolactinemia in women with anti-PRL autoantibodies.

Macroprolactinémie, une variété d'hyperprolactinémie. À propos de cinq observation

Macroprolactinemia, due to increased circulating levels of large molecular weight forms of prolactin, results in elevated level of immuno-reactive prolactin. The big variants have only weak biological activity; thus macroprolactinemia appears as a case of hyperprolactinemia without clinical significance as demonstrated by the five patients described. The diagnosis is based upon chromatography which separates the hormone and its variants. This disorder produces a pitfall in the diagnostic evaluation of hyperprolactinemia.

Urinary immunoreactive brain natriuretic peptide in patients with renal disease

Urinary immunoreactive brain natriuretic peptide (BNP) was studied by radioimmunoassay in patients with renal disease. Urinary immunoreactive human BNP excretion measured in 11 normal subjects was 3.82 ± 0.62 pmol/day (mean ± SEM). Significantly increased 24-h urinary secretion of immunoreactive human BNP was noted in patients with chronic renal failure (11.07 ± 1.73 pmol/day, n = 9, P < 0.05 to normal subjects). A significant correlation was noted between 24-h urinary excretion of immunoreactive human BNP and creatinine clearance in patients with various renal diseases ( r = −0.43, P < 0.01, n = 45). Gel chromatography of the urine extracts obtained from normal subjects and patients with chronic renal failure showed multiple immunoreactive peaks; two eluting earlier, one in the position of human BNP-32 and others eluting later. Reverse-phase high-performance liquid chromatography of the urine extracts showed a peak in the position of human BNP-32 and a peak eluting earlier. These findings indicate that: (1) immunoreactive human BNP is present in human urine; (2) urinary immunoreactive human BNP consists of multiple components, i.e., human BNP-32 itself or a substance very similar to it, smaller molecular forms which are probably metabolic products of human BNP-32, and larger molecular forms; and (3) 24-h urinary excretion of immunoreactive human BNP is increased in patients with renal dysfunction.

Urinary immunoreactive brain natriuretic peptide in patients with renal disease

Urinary immunoreactive brain natriuretic peptide (BNP) was studied by radioimmunoassay in patients with renal disease. Urinary immunoreactive human BNP excretion measured in 11 normal subjects was 3.82 ± 0.62 pmol/day (mean ± SEM). Significantly increased 24-h urinary secretion of immunoreactive human BNP was noted in patients with chronic renal failure (11.07 ± 1.73 pmol/day, n = 9, P < 0.05 to normal subjects). A significant correlation was noted between 24-h urinary excretion of immunoreactive human BNP and creatinine clearance in patients with various renal diseases ( r = −0.43, P < 0.01, n = 45). Gel chromatography of the urine extracts obtained from normal subjects and patients with chronic renal failure showed multiple immunoreactive peaks; two eluting earlier, one in the position of human BNP-32 and others eluting later. Reverse-phase high-performance liquid chromatography of the urine extracts showed a peak in the position of human BNP-32 and a peak eluting earlier. These findings indicate that: (1) immunoreactive human BNP is present in human urine; (2) urinary immunoreactive human BNP consists of multiple components, i.e., human BNP-32 itself or a substance very similar to it, smaller molecular forms which are probably metabolic products of human BNP-32, and larger molecular forms; and (3) 24-h urinary excretion of immunoreactive human BNP is increased in patients with renal dysfunction.

Development of Radioimmunoassay for Human Leptin

Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma human leptin-LI concentration in nonobese subjects (17.6 < body mass index (BMI) < 23.0) was 5.6 ± 1.3 (mean ± SE) ng/ml, while that in obese subjects (29.0 < BMI) was 43.0 ± 43.0 ± 9.4 (mean ± SE) ng/ml. In gel permeation chromatographic analyses, leptin-LI in culture media and plasma consisted of single component emerging at the elution position of recombinant human leptin. These findings indicate that leptin is secreted from the adipose tissue into the circulating blood as a large molecular form corresponding to recombinant leptin. The RIA developed in the present study will be a powerful tool to investigate the physiological and pathophysiological significance of leptin in human.

A high-affinity soluble folate receptor in fluids of non-neoplastic ovarian cysts: radioligand binding, molecular size, hydrophobic residue, and immunological properties.

The presence of a soluble folate receptor in fluids of non-neoplastic ovarian cysts was demonstrated. Radioligand binding exhibited characteristics typical of high-affinity folate-binding proteins. These included positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 and inhibition by folate analogues. The folate receptor was probably synthesized in the lining epithelial cells of the cysts which showed positive immunostaining with antibodies against human milk folate-binding protein. The gel filtration profile of cystic fluid contained two radioligand-bound peaks, 25 and 100 kDa, whereas a single band of 70 kDa was seen on SDS-PAGE immunoblotting. Treatment with the enzyme phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100 kDa peak to the 25 kDa peak. This suggests that insertion of a hydrophobic glycosylphosphatidylinositol tail into Triton X-100 micelles could give rise to large molecular size forms of the receptor on gel filtration.

Intravenously injected insulin-like growth factor (IGF) I/IGF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats.

Insulin-like growth factor (IGF) circulates in blood in two large molecular mass forms of 150 and 40 kD. Under normal conditions, most of the IGF is bound to the 150-kD complex by which it is retained in the circulation and therefore unable to exert acute insulin-like actions. The aim of this study was to answer the question whether or not IGF in the 40-kD complex is bioavailable to insulin target tissues and thus can cause acute insulin-like effects in vivo. Intravenously injected 1:1 molar recombinant human (rh) IGF I/rhIGF binding protein (BP)-3 complex lowered blood glucose and stimulated glycogen synthesis in diaphragm of hypophysectomized, but not of normal rats. The serum half-lives of the two components of the complex were similar to each other, but considerably shorter in hypox than in normal rats. On neutral gel filtration of serum both components of the injected complex appeared predominantly in the 150-kD region in normal rats. In hypox rats which lack the 150-kD complex they were found in the 40-kD region and disappeared rapidly from the circulation. We conclude that in the absence of the 150-kD complex, IGF associated with the 40-kD complex can rapidly leave the vascular compartment, reach insulin or type 1 IGF receptors and exert acute insulin-like effects.

A processing-independent assay for human procholecystokinin and its products

In order to develop a processing-independent analysis for procholecystokinin (proCCK) and its products, antibodies were raised against the synthetic fragment 62–71 of human proCCK. All rabbits ( n = 8) responded to the immunization. One (No. 89,009) produced antibodies of particularly high titer (1:350,000), homogeneity (Sips' index ~1.0) and binding affinity ( K o eff ~ 0.88 × 10 12 l/mol). A radioimmunoassy using this antiserum and [ 125I]tyrosine-extended fragment 62–71 measured the total CCK mRNA product after tryptic cleavage at Lys 61 in normal and neoplastic tissue independent of the degree of precursor processing. In addition to previously known CCK producing tumors, CCK was found also in a thoracic round-cell tumor (Askin tumor) and in brain tumors (gliomas and astrocytomas). These tumors processed proCCK poorly. Thus, they contained 11 and 23 (mean n = 5) pmol/g of proCCK and its products before, versus 71 and 99 (mean) pmol/g after tryptic cleavage, respectively. Accordingly, gel chromatography revealed significant amounts of unprocessed proCCK, large molecular forms of glycine-extended CCKs and the well-known carboxyamidated and tyrosine O-sulfated bioactive CCK-83, -58, -33, -22 and -8. We conclude that monospecific antibodies directed against the N-terminus of sequence 62–71 of human proCCK are suitable for processing-independent analysis (PIA) for proCCK and its products. Moreover, we suggest that such PIA should be used for quantitation of CCK gene expression at peptide level in normal tissue and tumors.

BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constituitively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M r and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Differential Influence of ATP on Native Spinach 70-Kilodalton Heat-Shock Cognates

A constitutively expressed class of 70-kD heat-shock cognate (HSC70) proteins from spinach leaf tissue was purified based on their affinity for ATP-agarose. The affinity-purified spinach proteins were resolved into at least three different forms on two-dimensional gels. Under native conditions, and iN the absence of ATP, the affinity-purified proteins were separated into three molecular mass classes by gel-filtration chromatography; a monomer of 85 kD, a multimer of 280 kD, and a large molecular mass oligomer of > 650 kD. All molecular mass forms contained a major protein that migrated at 79 kD on sodium dodecyl sulfate-polyacrylamide gels. N-terminal sequencing of the 79-kD purified monomer showed the highest homology to the endoplasmic reticulum-luminal HSC70. Addition of Mg-ATP to monomeric HSC70 did not alter its migration during gel filtration. Addition of Mg-ATP to the dimer converted it to monomer and oligomeric forms, whereas the presence of ATP converted a fraction of the large molecular mass oligomeric form of HSC70 to dimeric and monomeric forms. Only the large molecular mass oligomeric HSC70 appears to autophosphorylate in vitro in the presence of [gamma-32P]-ATP. Dimers and monomers can bind ATP by a nonhydrolytic mechanism and undergo a conformational change in the presence of Mg-ATP. In this paper we discuss the effects that ATP may have on the regulation of plant HSC70.

Unique change of pancreastatin-like immunoreactivity in cerebrospinal fluid by aging

Using a specific antiserum for the C-terminal glycine amide region of human pancreastatin (PST), pancreastatin-like immunoreactivity (PST-LI) was measured in cerebrospinal fluid (CSF) from 447 subjects (368 ± 10.8 pmol/l, mean ± S.E.M.) free from endocrine diseases. The CSF contents of PST-LI showed a mountain-shape type change which peaked at 40 years of age. The highest concentration was found in the group of ages 40–49 years old (412 ± 22.9 pmol/l) and the lowest concentration was found in the group of ages 80–89 years old (293.2 ± 45.2 pmol/l) among various age groups. Gel chromatographic examination revealed the presence of two major forms (MW 13,500 and 5,400) of PST-LI in CSF. Because of the character of this antibody, the large molecular form is possibly an N-terminally elongated PST and the other may be PST-52. This may be the first report on the unique age-related change of PST concentration in CSF.

Inhibition of pro-cholecystokinin (CCK) sulfation by treatment with sodium chlorate alters its processing and decreases cellular content and secretion of CCK 8

Pro-cholecystokinin (CCK) has three sulfated tyrosine residues. Sulfation of the tyrosine residue in CCK 8 is known to be important for its activity at CCK A receptors. The role of these sulfated tyrosines in the sorting and processing of pro-CCK was examined by treatment of CCK-secreting rat thyroid medullary carcinoma cells with 10 nM sodium chlorate (a non-toxic inhibitor of tyrosine sulfation). This treatment caused a 50% decrease in the cellular content of immunoreactive CCK and an 80% decrease in its secretion. Sephadex G-50 chromatography of cellular extracts and culture media showed a selective depletion of CCK 8. There was a comparative sparing of CCK 33 and larger molecular forms in cellular extracts which was not observed in the media. These results suggest that the sulfation of the tyrosines of pro-CCK is clearly important for the correct sorting and/or processing of pro-CCK. The pattern of immunoreactive CCK peptides seen with chlorate treatment is consistent with the substrate specificity of a recently identified putative CCK cleaving enzyme and suggests that unsulfated pro-CCK is not efficiently processed to CCK 8 in vivo. The large decrease in CCK content and secretion observed with sodium chlorate may also be due to inefficient sorting of unsulfated pro-CCK into secretory vesicles.

Clinical and biochemical effects in vivo of monoclonal antitumor antibody in verner-morrison's syndrome

Monoclonal antibodies have not been evaluated in metastasizing endocrine tumors, even though these lesions may induce severe morbidity of hormone excess in absence of considerable tumor burden. A murine monoclonal antibody of the IgG2a subtype was generated by immunization with dispersed tumor cells from an endocrine pancreatic carcinoma associated with liver and peritoneal metastases as well as a therapy-resistant Verner-Morrison's syndrome. Immunohistochemical staining disclosed selective tissue reactivity of the antibody and conspicuous immunostaining on the surface of the tumor cells. Infusion of 100 mg antibody over 2 days into the common hepatic artery of the patient was accompanied by reduced diarrhea volume until death 6 weeks later and transient elevation of total plasma immunoreactivity for vasoactive intestinal peptide due to large molecular forms of the peptide. Postmortem examination demonstrated disappearance of peritoneal metastases as well as absence of immunostaining for the injected antibody and the transferrin receptor within residual hepatic tumors. The results substantiate that symptomatic alleviation and perhaps interference with tumor cell mass may be obtained with monoclonal antibodies in patients with endocrine tumors and that the antiidiotypic immunoglobulin response may play a role herein.

Pancreastatin molecular forms in normal human plasma

Circulating molecular forms with pancreastatin (PST)-like immunoreactivity in plasma from normal subjects were examined. An immunoreactive form corresponding to a human PST-like sequence [human chromogranin-A-(250–301)] (hPST-52) and a larger form (mol wt 15–21 kDa) were detected by gel filtration of plasma from normal subjects. On high performance liquid chromatography, predominant immunoreactive forms coeluted with the three larger forms which were purified from the xenograft of human pancreatic islet cell carcinoma cell line QGP-1N cells and with synthetic hPST-52. The fraction containing larger forms purified from xenograft of QGP-1N cells had biological activity equivalent to that of hPST-52 on the inhibition of pancreatic exocrine secretion. These results suggest that the larger molecular forms as well as hPST-52 may be physiologically important circulating forms of PST in human.

Identification and Stabilization of Large Molecular Weight PDE-IVs from U937 Cells

Cytosolic cyclic nucleotide phosphodiesterases (PDEs) from human (promonocytic) U937 cells were rapidly resolved by DEAE-Sepharose CL-6B anion exchange chromatography into two major peaks of cAMP-specific activity possessing average Kms of 1.70 μM (Peak 1) and 1.65 μM (Peak 2). Both peaks were predominantly PDE-IV, but possessed molecular weights higher than those generally reported for partially purified PDE-IVs. Storage of Peak 2 for 24 h at 4°C resulted in a doubling of its Vmax and an apparent decrease in its molecular weight. Activation of Peak 2 PDE-IV was prevented when the sodium acetate concentration in its buffer was reduced by dilution immediately following isolation. Although the relevance of this activation to cellular regulation of PDE-IV is undefined, the isolation and stabilization of PDE-IV in its large molecular weight form will be critical to future investigations of PDE-IV regulation.

Enhanced expression of neurotensin/neuromedin N mRNA and products of NT/NMN precursor processing in neonatal rats

Intestinal levels of immunoreactive neurotensin (iNT) and neuromedin N (iNMN), as well as mRNA for the NT/NMN precursor, were elevated during the suckling period in rats. While transient expression of NT/NMN was observed at 1–5 days of age in the proximal small intestine and colon, NT/NMN levels in the ileum increased to peak at 10–20 days of age and then decreased to adult levels. The levels of these peptides were not elevated in the central nervous system and pituitary over this time period. Chromatographic analyses of jejunoileal extracts indicated that large molecular forms of iNT and iNMN were present, constituting ∼ 1.3% of total iNT and ∼ 56% of total iNMN, respectively. Treatment of the large forms with pepsin, which is known to generate the fully immunoreactive peptides, NT(3–13), NT(4–13), and NMN, increased immunoreactivity tenfold (iNT) and 1.2-fold (iNMN). Thus, large forms actually constituted ∼ 13% (iNT) and ∼60% (iNMN). Based upon its physicochemical properties, large molecular iNMN was tentatively identified as a 125 residue peptide with NMN at its C- terminus [i.e., rat prepro-NT/NMN(23–147)]. The properties of large molecular iNT were most similar to those predicted for the entire precursor [i.e., rat prepro-NT/NMN(23–169)]. These results indicate a) that enhanced expression of NT/NMN occurs in a tissue-specific manner in rats during the suckling period; b) that the pattern of precursor processing in intestine yields primarily NT and a large molecular form of NMN.