Hapten concentrations in the circulation of animals actively immunized with hapten-protein conjugates.

Animals immunized with hapten-protein conjugates subsequently circulate high concentrations of hapten bound by antibody. The levels of hapten detected are capable of significantly reducing antibody titer in the sera immunized animals. In the case of steroid-protein conjugates, the main source of increased plasma steroid concentration is the immunizing conjugate, although a contribution from increased host secretion may also occur. The results for rabbits immunized with digoxin-BSA indicate that the appearance of circulating digoxin followed the appearance of circulating antibody to digoxin. Appearance of digoxin in circulation appears to coincide with the operation of the immune response and may be related to macrophage activity. Similar conclusions are drawn from results obtained for circulating morphine in the serum of a sheep immunized with morphine-BSA. Injected hapten-protein antigens are probably processed by macrophage to produce low molecular weight haptenic fragments which are maintained in circulation for prolonged periods in the form of antibody-hapten complexes.     

Identification and preliminary characterization of two human digitalis-like substances that are structurally related to digoxin and ouabain.

In order to characterize the structure of endogenous digitalis-like immunoreactive factor (DLIF), we utilized peritoneal dialysis fluid from patients with chronic renal failure as a source of endogenous digitalis-like immunoreactive factor (DLIF), and subjected it to one-step ion exchange chromatography, followed by one step reverse HPLC. Crude dialysis fluid contained 0.09 ng/ml of DLIF, and using Amberlite XAD-2 chromatography we extracted 110 ng of DLIF from 800 ml of dialysis fluid. By applying this partially purified DLIF to our HPLC system, we discerned three peaks of DLIF activity, with retention times of 34, 58 and 63 minutes. The first peak overlapped the elution profile of ouabain, and the third peak co-eluted precisely with digoxin. The second DLIF peak was not in proximity to any of the digitalis-like markers employed. Thus, our results indicate that DLIF isolated from peritoneal dialysis fluid exists in three distinct forms, one of which resembles ouabain, and one which is identical to digoxin.     

Digoxin-like immunoreactive factor in rat plasma: effect of sodium and calcium intake

Studies have been performed in rats to determine whether an endogenous material capable of binding to digoxin antibodies is present in the plasma. Such a material has been shown in other species and has been hypothesized to represent an endogenous ligand for the receptor on Na-K ATPase through which cardiac glycosides act. In rats consuming a normal rodent chow (1% calcium by weight) and drinking deionized water, endogenous binding of digoxin antibody in radioimmunoassay amounted to 23.1 +/- 4.6 fM digoxin equivalents/100 microliter of plasma (mean +/- SEM, n = 8). Since a hypothetical role for such an endogenous ligand is the regulation or renal sodium excretion by inhibition of renal Na-K ATPase, the effect of increased sodium intake on plasma levels of this digoxin-like immunoreactive factor (DLIF) was studied. Animals consuming the same chow, but drinking 0.5% NaCl solution in place of water for a 4 week period showed significantly greater DLIF in plasma which was measured at 109.2 +/- 20.3 fM digoxin equivalents/100 microliter of plasma (p less than 0.001). Because DLIF has been implicated in the pathogenesis of hypertension we also studied the effects of calcium intake on plasma levels of DLIF. In previous studies we have shown that rats allowed to drink 0.5% saline develop a moderate hypertension which can be reversed with calcium supplementation. In the present studies, 3 dietary calcium subgroups (0.01% Ca, 1.0% Ca and 4% Ca) were formed among animals drinking water or 0.5% saline for 4 weeks. No effect of low calcium intake on plasma DLIF was found either in water or saline drinkers. However, calcium supplementation produced a significant reduction in plasma DLIF in both water and saline drinking animals.     

Critical examination for the presence of a low molecular weight fraction in serum iron

Native human pool serum and individual sera were ultrafiltered by Pellicon ultrafilters (Millipore) and the ultrafiltrates were extracted by an ammonium pyrrolidinedithicarbamate/methylisobutylketone system after addition of different internal iron standards to three of four identical ultrafiltrates. The extracts were examined for iron content by atomic absorption spectrometry. During ultrafiltration pH 7.4 was miantained by a constant atmosphere of a CO₂/air mixture.Low molecular weigth iron in native human sera from normal, normal orally iron substituted and siderotic individuals was found to be less than 0.05 μg/100 ml. Elevating serum citrate to 3-fold normal had no effect on this result.More iron became ultrafiltrable if the serum pH were lowered by citric acid as compared with hydrochloric acid.     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

Differential effect of fasting on IGF-BPs in serum of young and adult rats and its implication to impaired skin GAG content

During fasting or aging of animals there is a decreased content of skin glycosaminoglycans (GAGs). It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals (young and adult) resulted in decrease of GAG content. However, GAG content in the skin of fasted young rats decreased by 30% and in fasted adult rats by 15% only, compared to fed animals, respectively. The mechanism for the phenomena is not known. We considered insulin-like growth factor-I (IGF-I) as a potential candidate involved in regulation of GAG biosynthesis in both experimental models of animals. Adult rat sera were found to contain about 75% of IGF-I recovered from young rat sera. Fasting of both groups of animals resulted in dramatic decrease in serum IGF-I levels to about 50% of initial values. Since IGF-I activity and IGF-I serum half-life depends on the level of specific IGF-binding proteins (IGFBPs) we determined (i) relationship between main groups of IGFBPs, namely high molecular weight binding proteins (HMWBPs) and low molecular weight binding proteins (LMWBPs) and (ii) the amounts of IGF-I bound to respective proteins in the sera of all experimental animals. Control young rat serum was found to contain about 90% of HMWBPs and about 10% of LMWBPs as determined by ligand binding assay. In contrast, control adult rat serum contained about 60% of HMWBPs and about 40% of LMWBPs. Fasting of both groups of animals resulted in significant increase in serum levels of LMWBPs. Control young rat serum was found to contain about 8% IGF-I bound to LMWBPs while serum of control adult rats contained 18% IGF-I bound to these proteins. In sera of fasted young animals however, about 75% of the bound IGF-I was recovered from LMWBPs (about 60% of total serum IGF-I) while in sera of fasted adult animals only about 56% of the bound IGF-I was recovered from LMWBPs (about 50% of total serum IGF-I). Evidence was provided that during fasting of both groups of animals there is a significant decrease in serum BP-3 and dramatic increase in serum BP-1 concentrations, compared to respective controls. However, the concentration of BP-1 in serum of fasted young rats was increased by about 60 fold while in serum of fasted adult rats only by about 10 fold, compared to respective control animals. Negative correlation between skin GAG content and LMWBPs derived IGF-I during fasting of young (r = - 0.943, p < 0.001) and adult ( r = - 0.571, p < 0.01) rats was found.The data presented suggest that the effects of aging and fasting on decreased skin GAG content may be due to induction of LMWBPs that are known to (i) inhibit IGF-I dependent function and (ii) increase clearance of IGF-I from circulation. However, the effects of fasting are distinct in respect to young and adult rats suggesting that mechanisms involved in regulation of IGF-I bioactivity during aging are more complex that during fasting.     

Isolation of digoxin-like immunoreactive factors from mammalian adrenal cortex

Endogenous digoxin-like immunoreactive factors (DLIF) are present in serum and tissues of humans and animals. To date, a tissue source for these factors has not been rigorously defined nor have these factors been isolated to identifiable homogeneity. In this study, we define the distribution of DLIF in mammalian tissues, demonstrate the adrenal cortex to be the principal source of this factor in bovine, and isolate DLIF to chromatographic homogeneity using high performance liquid chromatography (HPLC). DLIF concentrations in tissue extracts from rats measured as follows: adrenal glands, 44.3; serum, 6.3; liver, 5.2; kidney, 1.2; heart, brain, or lungs, less than 1.4 ng of digoxin-equivalent per g of protein. Human tissues showed similar results. In dogs, the ratio of the DLIF concentration in lumbar vein serum to that in infrarenal inferior vena cava serum was 3.3 +/- 0.4 (mean +/- S.E., n = 4). Bovine adrenal cortex contained 7 times more DLIF per g of tissue than the adrenal medulla. 70 +/- 4% (n = 7) of the total bovine cortical DLIF activity (6,159 pg of digoxin-equivalent) applied to a reverse phase HPLC column eluted as one definitive fraction. 60% of the digoxin-like immunoreactivity extracted from bovine serum also co-eluted with DLIF from adrenal. None of the 14 steroid molecules or 7 cardiac glycoside congeners co-eluted with the major DLIF activity. Our data indicate that 947 pmol of DLIF is equivalent to 1 pmol of digoxin-equivalent immunoreactivity. Preliminary mass spectral analysis suggests that purified DLIF has a molecular mass of 780 daltons comprised of one 390-dalton aglycone component plus several sugar moieties. This study establishes a definitive link between DLIF in serum and the adrenal cortex as a source tissue. We also demonstrate a method for purifying DLIF to chromatographic homogeneity with an extraction capacity of 1.2 nmol of DLIF per g of adrenal cortex.     

Ribosomal proteins

Summary Ribosomal proteins of E. coli and yeast were separated by gel filtration on dextran (Sephadex) and polyacrylamide (Bio-Gel) columns. Both gels revealed a valuable separation of the proteins. Finally only Bio-Gel columns were used, since their polyacrylamide matrix is more resistant to the applied organic acids.The wide distribution of the molecular weights for both the E. coli and yeast ribosomal proteins was confirmed. E. coli ribosomal proteins were separated into three main groups by a single chromatography on Bio-Gel P-10. The same was true for yeast ribosomal proteins. Rechromatography of these protein groups resulted in a further valuable resolution. The fractionated proteins are recovered without any loss and they are very useful for further purification by other procedures, especially on a large scale basis.     

Age-related changes in the growth-stimulating effect of serum on BHK-21 cells

The growth-stimulating effect of two calf and adult animal serum ultrafiltrates (with molecular weight of the components up to 100 000 dalton) on BHK-21 cells has been compared. The growth-stimulating activity of the calf serum ultrafiltrate was much greater. Aggregation of cells--the result of their reduced adhesion to the substrate--was much less pronounced in the presence of the calf serum ultrafiltrate than in the medium with the serum ultrafiltrate of adult animals. The data obtained show that the components containing in the studied ultrafiltrates (mol/wt up to 100 000 dalton) and not gamma-globulins (mol/wt 150 000 dalton), as it was considered earlier, primarily determine the ageing change of growth-stimulating activity of the serum. It should be suggested that the different influence of ultrafiltrates on cell adhesion is due to the presence of factors of adhesion with molecular weight up to 100 000 dalton in the calf serum and to its absence in the serum of adult animals.     

A simple method for xylanase preparation used for the hydrolysis and fermentation of hemicellulose to butanediol

Summary When culture filtrates ofTrichoderma harzianum E58 were concentrated by passage through an ultrafiltration membrane with a molecular weight exclusion limit of 10,000 Daltons, 80% of the original xylanase activity was recovered in the ultrafiltrates. Culture filtrates and ultrafiltrates which were concentrated by rotary evaporation contained inhibitors which restricted the fermentation of the hemicellulose-derived sugars to 2,3-butanediol. A simple solvent-exchange treatment of the ultrafiltrates could effectively concentrate the xylanases as well as remove the fermentation inhibitors.     

Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalo-virus (CMV) infection, B₁₂-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (>1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation.The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity.The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.     

Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

Molecular characterization of {beta}-trace protein in human serum and urine: a potential diagnostic marker for renal diseases

We have isolated ß-trace protein from cerebrospinalfluid, serum, plasma, and urine samples of normal volunteersand sera and hemofiltrate of patients with chronic renal failure.Blood-derived and urinary ß-trace have significantlyhigher molecular weights than their cerebrospinal fluid counterpart,the amino acid sequences being identical. Oligosaccharide structuralanalysis revealed these molecular weight differences to be dueto different N-glycosylation. ß-Trace from hemofiltrateand urine has larger sugar chains and concurrently significantlyhigher sialylation than cerebrospinal fluid-ß-tracewhich bears truncated "brain-type" oligosaccharide chains (publishedpreviously). ß-Trace concentrations were about 40ng/ml for normal sera and plasma. 2000–6000 ng/ml weremeasured in sera of dialysis patients whereas in normal humancerebrospinal fluid, ß-trace concentration was about8000 ng/ml. A reduced amount of 900 ng/ml was found in a singlecase of hydrocephalus cerebri. The sialylated glycoforms ofß-trace detected in the blood are presumably derivedfrom resorbed cerebrospinal fluid protein whereas ß-TP-mole-culesbearing asialo-oligosaccharides are absent due to their hepaticclearance. The residual, sialylated ß-TP-species areprobably eliminated from the blood via the kidney. This physiologicalclearance mechanism for the sialylated glycoforms is disturbedin hemodialysis patients resulting in about 100-fold elevatedserum concentrations. These results let us suggest ß-tracemay become a useful novel diagnostic protein in renal diseases. "brain-type" N-glycosylation hepatic clearance human ß-trace kidney failure serum glycoproteins     

Effect of ultrafiltrates in plasma from patients with chronic uremia on platelet aggregation]

An effect of 20 ultrafiltrates from patients with chronic renal failure on ADP-, epinephrine-, and arachidonic acid-induced platelet aggregation. Ultrafiltrates were collected at the beginning and the end of dialysis. It was found that ultrafiltrates collected at the beginning of dialysis decrease ADP-induced platelet aggregation whereas ultrafiltrates collected at the and of dialysis exert reverse effect. No effect on epinephrine- and arachidonic acid-induced platelet aggregation was seen. Removal of substances inhibiting platelet aggregation produced by ADP hemodialysis may reveal increased platelet aggregation in patients with uremia.     

Comparative immunochemistry of phytochrome

Partially purified high molecular weight preparations of phytochrome, estimated to be close to 440,000 molecular weight based upon chromatography through a calibrated Bio-Gel P-300 column, were obtained from Garry and Newton oats (Avena Sativa L., cv. Garry and cv. Newton), rye (Secale cereale L., cv. Balbo), barley (Horedum vulgare L., cv. Harrison), and pea (Pisum sativum L., cv. Alaska) by a sequence of three chromatographic steps: brushite, diethylaminoethyl cellulose, and Bio-Gel P-300. No significant differences were observed between these preparations during purification or subsequent handling. In addition, a low molecular weight form of phytochrome was purified from Garry oats. Two specific antisera against a low molecular weight form of phytochrome (60,000 molecular weight) obtained from etiolated Garry oat seedlings are characterized and used to compare the phytochrome preparations. Double diffusion assays indicated antigenic identity between all preparations except that pea phytochrome yielded a spur when compared to oat phytochrome. Micro complement fixation assays yielded complete identity between Garry and Newton oat phytochrome, reduced activity with rye and barley phytochrome, and a complete lack of activity with pea phytochrome at the serum dilutions assayed. Immunoelectrophoretic assays indicated that all high molecular weight phytochrome preparations were homogeneous by this criterion and that there were only slight differences between the preparations in electrophoretic mobility. Large and small forms of phytochrome isolated from Garry oats were found to be very similar antigens when tested with the anti-small phytochrome sera, although the small form was observed to electrophorese at a much slower rate than the large.     

Calcification inhibitors in rat and human serum and plasma

Rat and human serum and plasma were shown to contain considerable amounts of calcium phosphate precipitation inhibitors. Two general classes of inhibiting molecules were observed for both species: high molecular weight (approx. 30 000-200 000) and low molecular weight (less than 1000). The high molecular weight components eluted from a Bio-Gel P-200 column in two peaks, one at approx. 158 000 and a broader peak at approx. 43 000. The identity of these inhibitors is unknown at present. Low molecular weight inhibitors include magnesium, pyrophosphate, and citrate ions and at least one unidentified component that coelutes with pyrophosphate and citrate on a Bio-Gel P-4 column. Quantitatively, most of the inhibitor activity resides in the high molecular weight components and it is possible that it is this activity which is responsible for maintaining the metastability of the circulating fluids. The role of the low molecular weight components may be to regulate calcification at sites inaccessible to high molecular weight molecules.