Degradation of serum amyloid A and apolipoproteins by serum proteases

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.     

Radioimmunoassay of haloperidol in human serum: Correlation of serum haloperidol with serum prolactin

A radioimmunoassay (RIA) for measurement of serum haloperidol is described. Compared to gaschromatography (GC), RIA values average 40% higher. However, a simple organic extraction of serum yields statistically equivalent RIA and GC haloperidol determinations. For both men and women combined, there was a positive correlation between dose (mg/kg/day) and steady-state serum haloperidol level (r = +0.86) and between steady-state serum haloperidol and serum prolactin (PRL) concentration (r = +0.87).     

Urinary and serum titanium

Ammonium citratoperoxotitanate IV (TAS-FINE) is a water-soluble titanium complex used to synthesize a photocatalytic titanium(IV) oxide film. This study was aimed to investigate the LD₅₀, dose-response, time-course response, and renal toxicity of TAS-FINE using an animal model. Serum titanium (S-Ti) and its 24-h urinary excretion (U-Ti) were determined by inductively coupled plasma-argon emission spectrometry (ICP-AES) after a single oral TAS-FINE administration to male Wistar rats. The LD₅₀ of TAS-FINE was 7.97 g/kg body weight in 24 h, and its half-life was 3.78±1.28 d for S-Ti and 2.19±0.09 d for U-Ti. Although TAS-FINE was not easily absorbed in the gastrointestinal tract, it was distributed into the bloodstream in a dose-dependent manner. Within 24 h, 0.189% of administrated Ti was excreted via urine. It was speculated that TAS-FINE formed conjugates with serum constituents that resulted in nephrotoxicity resulting from an allergic reaction. The observed indices in this study were revealed to be good indicators for TAS-FINE exposure. The analytical method and animal model described in this study will help to further elucidate details about human exposure to TAS-FINE, which in recent times has become an occupational and environmental toxicant of concern.     

Antileptospiral activity of serum. I. Normal and immune serum

Johnson, Russell C. (University of Minnesota, Minneapolis), and Louis H. Muschel. Antileptospiral activity of serum. I. Normal and immune serum. J. Bacteriol. 91:1403-1409. 1966.-Normal serum was found to exert a leptospiricidal effect, mediated by the complement system, against the nonpathogenic leptospires. Although resistant to normal serum, the pathogenic serotypes were susceptible to antiserum plus complement. Several variables in these immune leptospiricidal reactions were investigated. A reaction period of 3 hr at 37 C between serum substances and 1-day-old cells provided a maximal leptospiricidal effect. The normal serum of the rabbit, guinea pig, bovine, and human were leptospiricidal against the nonpathogenic serotypes, and, in conjunction with rabbit antiserum, rabbit and bovine complement were leptospiricidal against the pathogenic serotypes. Studies with C(14)-labeled leptospires indicated that the immune leptospiricidal reaction was associated with a loss of permeability control. Thus, like the gram-negative bacteria, the treponemes, erythrocytes, and nucleated mammalian cells, the leptospires may be included as cell types susceptible to the antibody-complement system.     

Effects of whole-body cryotherapy on serum mediators of inflammation and serum muscle enzymes in athletes

Whole-body cryotherapy (WBC) covers a wide range of therapeutic applications and consists of briefly exposing the body to extremely cold air. In sports medicine, WBC is used to improve recovery from muscle injury; however, empirical studies on its application to this area are lacking. To fill this gap, we compared changes in immunological parameters (C3, IgA, IgM, IgG, C-reactive protein, PGE2), cytokines (IL-2, IL-8, IL-10), adhesion molecules (sICAM-1), and muscle enzymes (creatine kinase [CK], lactate dehydrogenase [LAD]) before and after WBC in 10 top-level Italian National team rugby players. The subjects underwent five sessions on alternate days once daily for 1 week. During the study period, the training workload was the same as that of the previous weeks. Compared to baseline values, immunological parameters remained unchanged, while CK and LAD levels significantly decreased after treatment. No alterations in immunological function were observed but there is a decrease in pro-inflammatory cytokine/chemokine and an increase in anti-inflammatory cytokine.As measured by changes in serum CK and LAD concentrations, and cytokines pathway, short-term cold air exposure was found to improve recovery from exercise-induced muscle injury and/or damage associated with intense physical training.     

Aggregation and fibrillation of bovine serum albumin

The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a lag phase, is independent of seeding and shows only moderate concentration dependence, suggesting intramolecular aggregation nuclei. Nevertheless, multi-exponential increases in dye-binding signal and changes in morphology suggest the existence of different aggregate species. Although beta-sheet content increases from 0 to ca. 40% upon aggregation, the aggregates retain significant amounts of alpha-helix structure, and lack a protease-resistant core. Thus BSA is able to form well-ordered beta-sheet rich aggregates which nevertheless do not possess the same structural rigidity as classical fibrils. The aggregates do not permeabilize synthetic membranes and are not cytotoxic. The ease with which a multidomain all-alpha helix protein can form higher-order beta-sheet structure, while retaining significant amounts of alpha-helix, highlights the universality of the fibrillation mechanism. However, the presence of non-beta-sheet structure may influence the final fibrillar structure and could be a key component in aggregated BSA's lack of cytotoxicity.     

Effects of gonadotrophin-induced elevation of serum testosterone upon somatomedin C levels and serum thymidine activity in children

3H-thymidine uptake into lectin-activated human lymphocytes allows to measure a growth-stimulating activity of serum, the thymidine activity (TA), which is GH dependent in vivo and related to somatomedins (Sm). In this work, it is shown: that addition of human chorionic gonadotrophin (HCG) or testosterone in vitro does not increase the 3H-thymidine uptake into lymphocytes; that the gonadotrophin-induced elevation of testosterone in children is accompanied by a significant increase of TA and, at a lesser degree, of Sm C; that these two increases are significantly correlated, and that the age-related variation of TA and Sm C after HCG stimulation test are not parallel.     

Purification and characterization of human serum biotinidase

Biotinidase has been purified from human serum to a specific activity of 1900 units/mg protein by a five-step procedure. After ammonium sulfate precipitation (33-55% cut) it was purified by DEAE-Sephacel, hydroxylapatite, octyl-Sepharose CL-4B, and Sephadex G-100 chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions. Biotinidase is a glycoprotein. The sialic acid residues in the molecule are not required for enzyme activity. The Mr of human serum biotinidase estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ferguson plot) and by sedimentation analysis was 68,000. Human serum biotinidase showed maximum activity in the pH range 6.0 to 7.5 with N-(d-biotinyl) p-aminobenzoate as substrate. However, with biocytin as substrate, the maximal activity of the enzyme was in the pH range 4.5 to 6.0. Using structural analogs of the substrate we have shown that biotinidase is not a general proteolytic enzyme and has specific structural requirements in the substrate for hydrolysis.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Nongradient blue native gel analysis of serum proteins and in-gel detection of serum esterase activities

The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either α-naphthylacetate or β-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.     

The effect of thymus extracts on phosphorus compounds in muscle and serum, and on serum calcium

1. Thymectomy in young rabbits decreased the ATP content and increased the inorganic phosphate content of skeletal muscle. The serum calcium content was decreased, whereas the inorganic phosphate content was increased. 2. The administration of a lipid fraction (TL) or protein fractions (CIF and TP) of thymus extracts to thymectomized rabbits in short-term experiments increased the ATP content of muscle and decreased the inorganic phosphate contents of muscle and serum. Serum calcium content was increased. 3. The action of the thymus extract TP was specific only on the phosphate compounds, since the increase in serum calcium concentration was also caused by the control extract from muscle. The action of the extract TL is not specific, being paralleled by the action of a control extract from muscle.