Calmodulin content and distribution in six human melanoma cell lines

Calmodulin content and distribution between soluble and particulate fractions were determined by radioimmunoassay in six human melanoma cell lines exhibiting differences in tumor origin (primary or metastatic), degree of tumorigenicity and of pigmentation (amelanotic or melanotic). The results indicate that a) total, soluble and particulate calmodulin levels expressed as ng/10(6) cells or ng/micrograms of proteins remained constant for five out of six cell lines when cells grew from subconfluency to confluency. For IGR 37 line, derived from metastatic melanoma, the calmodulin content decreases from 2.39 to 1.27 ng/micrograms protein for total calmodulin, from 2.17 to 1.52 ng/micrograms protein for soluble calmodulin and from 2.61 to 1.02 ng/micrograms protein for particulate calmodulin, b) total, soluble and particulate calmodulin levels expressed as ng/microgram proteins were twofold (at confluency) to fourfold (at subconfluency) higher in the two cell lines from metastatic origin, IGR 37 and IPC 167. As for example, for total calmodulin, values in IGR 37 and IPC 167 cell lines, were, respectively at subconfluency, 2.39 and 2.31 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.96 ng/micrograms protein and at confluency: 1.27 and 1.98 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.90 ng/micrograms protein, c) ratio of calmodulin between soluble and particulate fractions was about 1 for the two autologous cell lines IGR 37 and IGR 39 and varies from 2 to 3 for the four other cell lines.     

A quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and 125I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and 125I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous 125I immunobinding assays which were not quantitative but were used as antigen "spot tests" or which required iodination of the antibody.     

Radioimmunoassay for Central Nervous System Myelin-Specific Proteolipid Protein

A double-antibody radioimmunoassay (RIA) has been developed with antisera to purified rat brain myelin proteolipid protein (PLP). The addition of Triton X-100 allowed antibody-antigen interaction and immune precipitation in the presence of sodium dodecyl sulfate (SDS). The RIA will accurately measure 8-80 ng of PLP in buffer or human serum. The RIA is highly specific for myelin PLP and does not cross-react with material in tissues (heart, kidney, muscle, testicle, and intestine) other than the central nervous system. The antibodies to rat myelin PLP cross-react with PLP from bovine brain homogenate or myelin. Myelin PLP was found to account for 55 and 52% of total myelin protein from bovine and rat brain, respectively. Furthermore, there is a higher concentration of PLP in white than in gray matter corresponding to the degree of myelination. Unlike myelin basic protein, myelin PLP was undetectable in both bovine and rat peripheral nervous system.     

Calmodulin-dependent protein phosphatase: a developmental study

Calmodulin-dependent protein phosphatase, one of the major calmodulin-binding proteins in bovine brain, dephosphorylates casein with a specific activity of 15 nmol mg-1 min-1 at 30 degrees C. The stimulation of phosphatase activity by calmodulin is reversed by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or trifluoperazine, a calmodulin antagonist. Antibodies raised in rabbit against the phosphatase inhibit the enzyme activity. The levels of the protein in brain extracts from various animals, determined by a radioimmunoassay, range from 20 micrograms/g of tissue in chick and fish brains to 143 micrograms in rat cerebrum. The ontogeny of the phosphatase was studied in nervous tissues from rat and chick, animals in which synaptogenesis takes place at different times during their development. The levels of the protein increased significantly in rat cerebrum and cerebellum and in chick brain and retina during the periods corresponding to major synapse formation. In rat cerebrum, the enzyme appeared to be equally distributed between the cytosol and the particulate fraction; the level in both compartments increased during the major period of synapse formation. Thus, the development of calmodulin-dependent protein phosphatase closely parallels synaptogenesis, implicating a role in some synaptic function.     

A Radioimmunoassay for the Phosphoprotein B-50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.     

P-57 is a neural specific calmodulin-binding protein

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.     

Identification of type III protein kinase C in bovine aortic tissue

We identified a subtype of protein kinase C in bovine aortic tissue. In Western blots, both the soluble and the particulate fractions from the aorta reacted only with MC-3a. In the case of hydroxylapatite column chromatography, a single activity peak of protein kinase C from the soluble and the particulate fractions was obtained with about 140 mM of potassium phosphate, a finding similar to that with the Type III protein kinase C from rabbit brain. The sandwich-type enzyme immunoassay for protein kinase C, with which the contents of each protein kinase C isozyme can be determined in the crude extracts, revealed that the Type III bovine aortic protein kinase C included 25.9 ng/mg protein. These results strongly suggest that it is the Type III protein kinase C which is mainly expressed in aortic tissue. Kinetic parameters of the Type III protein kinase C of the soluble and the particulate fractions, with respect to the Km for ATP, were 33 and 15 microM and the Km values for myosin light chain from chicken gizzard were 6.3 and 4.6 microM, respectively.     

Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: purification of a distinct cGMP-stimulated isoenzyme

In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat epidermal growth factors: purification and tissue content

Rat epidermal growth factor (rEGF) was isolated from the submaxillary gland of male rat by reversed phase high performance liquid chromatography and ion exchange chromatography. The binding of purified rEGF to human carcinoma cells (A-431) and its tritiated thymidine uptake on rat epidermal fibroblast cells (FR) were almost the same as those of purified commercially available mouse EGF (mEGF). Antisera to rEGF was raised in rabbits and a radioimmunoassay (RIA) system was established. The assay range of the RIA was about 1.0 to 100 ng/ml. The within assay coefficients of variation were 5 to 10%, while the between assay coefficients of variation were 5 to 13%. The tissue content of rEGF of male rats (10 weeks old) was examined. As a result, the submaxillary gland was found to contain a very high concentration of rEGF (214 micrograms/g wet tissue) as predicted, and digestive tissues, stomach, intestine and duodenum contained 2.49, 3.57, 9.44 ng/g wet tissue, respectively. The amounts in prostate and seminal vesicle were relatively high, being 65.6 and 2,268 ng/g wet tissue, respectively. The amount in the submaxillary gland increased markedly after 7 weeks of age. These results suggest that EGF is an important factor in gonadal function.     

Determination of metallothionein in tissues by radioimmunoassay and by cadmium saturation method

The diversity of reported basal metallothionein (MT) values in animal tissues has made it necessary that the presently available methods be further developed and compared for their accuracy, reproducibility, sensitivity, and specificity. A radioimmunoassay (RIA) to quantitate MT in animal tissues was developed and its performance compared to that of a cadmium saturation method. The procedure is more accurate and reliable but no more time-consuming than other techniques in current use. It offers the advantages of greater specificity and sensitivity thus enabling the determination of basal levels of MT in tissues and the analysis of small samples, for example, biopsies, cultured cells, in vitro protein synthesis, etc. The use of a polyclonal antiserum is advantageous in that total MT can be determined in any tissue from a variety of animal species. Both nonspecific and specific interference in the assay can be eliminated by heat treatment of the sample followed by a short preincubation with cadmium. The sensitivity of the RIA is 10 ng MT/g tissue. The cadmium saturation assay is unsuitable for the measurement of low levels of MT due to its nonspecific nature and its detection limit (10 micrograms MT/g tissue) but it is useful where large amounts of the protein are present in a tissue. Difficulties arising in the analysis of MT by both methods are discussed and solutions are offered. The basal levels of MT in the brain, kidney, liver, lung, muscle, pancreas, small intestine, and spleen of rats are determined by RIA and are shown to be generally lower than the currently accepted values measured by other techniques.     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of pancreatic polypeptide-like immunoreactivity in rat tissues

The distribution of pancreatic polypeptide (PP)-like immunoreactivity (LI) in rat tissue was determined by a specific radioimmunoassay (RIA) after extraction with boiling 1 N acetic acid. The concentration of PP-LI in the ventral area of the pancreas (0.917 +/- 0.106 micrograms/g tissue) was about 10 times greater than that in the dorsal area of the pancreas (0.085 +/- 0.006 micrograms/g tissue). Extrapancreatic PP-LI was present in the colon (0.034 +/- 0.010 micrograms/g tissue) and rectum (0.019 +/- 0.001 micrograms/g tissue). The remainder of the gastrointestinal tract, the lung, kidney, liver, spleen, heart, adrenal gland, and central nervous system contained no measurable PP-LI. Reverse-phase high performance liquid chromatography analysis of the PP-LI materials from the pancreas, colon, and rectum revealed one peak which corresponds to the rat PP standard, under conditions of elution which clearly separated PP, NPY, PYY. These results show that distribution of PP-LI in the rat is different from other known distributions in the PP family of peptides.     

Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay

A simple procedure for the determination of cellular proteins in Percoll-containing samples is described. Percoll precipitated when particulate proteins were solubilized by dilution of the samples in a NaOH-Triton X-100 mixture. After centrifugation at high speed (12,000g), the supernatant was assayed for proteins with the Coomassie brilliant blue dye-binding assay. With an automatic spectrophotometer, 50-microliter aliquots gave a linear response between 0 and 3 micrograms of bovine serum albumin. After a fivefold dilution in the alkali-detergent mixture, proteins in samples containing up to at least 60% Percoll can be accurately quantitated on a standard curve prepared in the absence of Percoll. Because the sensitivity of the assay was better than 100 ng, the procedure outlined in this paper can also be used as a general protein micromethod.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

Comparison of calregulins from vertebrate livers.

Calregulins were purified from bovine, rabbit and chicken liver, and their structural properties were compared. Significant differences between the three calregulins include a lower Mr for chicken calregulin (57,000) than for rabbit and bovine calregulin (63,000), and the glycosylation of only bovine calregulin. Amino acid composition and peptide maps of the three calregulins were very similar. No major differences were detected in the Ca2+-binding properties of the three proteins. Zn2+-induced changes in calregulin conformation and hydrophobicity monitored by intrinsic protein fluorescence and the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulphonate were very similar, suggesting that the Zn2+-dependent increase in the hydrophobicity of bovine, rabbit and chicken calregulin was conserved. These studies more fully define what is a calregulin, demonstrate that calregulin is a relatively invariant constituent of vertebrate liver, and indicate that calregulin structure has been highly conserved in bovine, chicken and rabbit liver.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Culture of transformed horseradish roots as a source of fusicoccin-like ligands

Endogenous fusicoccin (FC) or related substances were sought in horseradish (Armoracia rusticana P.) roots. An actively growing root culture was derived from plants transformed with Agrobacterium rhizogenes. The presence of FC-like substances in ethanolic extracts from roots was established in a radioreceptor binding assay with plasmalemmal FC receptors and in radioimmune analysis with an antiserum specific for FC A. FC-like ligands were found in the tissue and medium of aseptically grown culture.Abbreviations FC fusicoccin - GC/MS gas chromatography/mass spectrometry - RIA radioimmunoassay - RRA radioreceptor analysis - BSA bovine serum albumin - Mes 4-morpholineethanesulfonic acid - HPLC high performance liquid chromatography     

A two-site enzyme-linked immunosorbent assay for inhibin.

The investigation of the role of inhibin in the regulation of fertility is hindered by the lack of a routine, specific assay. The pituitary cell bioassay is time-consuming and the existing RIAs, based on either purified bovine 32-kDa inhibin or synthetic alpha-subunit peptides, are not specific for the biologically active inhibin molecules. We have used monoclonal antibodies, one specific for the N-terminal region of the human inhibin alpha chain, and the other raised to a peptide sequence close to the C-terminal of the human beta A-inhibin chain, to create a two-site sandwich ELISA specific for alpha beta-inhibin molecules. This was used to estimate levels of inhibin in crude bovine and human follicular fluids and fractions concentrated from them. Comparison of the values obtained with the ELISA and those obtained with the pituitary cell bioassay, suggests that the ELISA measures biologically active inhibin. Compared with the peptide-based RIA, the ELISA gave much lower (as little as 100-fold lower) values for the inhibin content of these samples, e.g., bovine follicular fluid 0.375 micrograms/ml (ELISA) compared with 41.0 micrograms/ml (RIA). Such large differences, possibly due to the presence of relatively large amounts of biologically inactive forms of inhibin such as the pro-alpha c or free alpha forms, suggest that those RIAs, which essentially measure the level of alpha-inhibin, considerably overestimate the levels of the active forms of inhibin in the samples and that results obtained using these assays may need reinterpretation.