Elevation of tissue coenzyme Q (ubiquinone) and cytochrome c concentrations by endurance exercise in the rat

Six months of enforced and voluntary endurance training of young female Wistar rats resulted in significant decreases of body weight and gastrocnemius muscle wet weight and protein content, and increases in heart weight and protein content, and liver protein content. The coenzyme Q and cytochrome c concentrations of cardiac, gastrocnemius, and deep red region of the vastus lateralis muscles were increased, while small or nonsignificant trends toward increases in cytochrome c and coenzyme Q were seen in kidney, brain, lung, liver, internal + external oblique muscles, and the superficial white region of the vastus lateralis muscle. These results are discussed with regard to several roles for coenzyme Q in cellular function.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: II. Characterization of mononuclear phagocytes extracted from rat cardiac allografts

A method was developed for the extraction of leukocytes infiltrating rat cardiac allografts. Mononuclear phagocytes (MNP) comprised 52.4 ± 5.5% of the cells extracted from allografts at the time of rejection (Day 7). Day 4 allografts and Day 7 syngeneic grafts yielded considerably fewer MNP although numbers of lymphoid cells were similar in all three groups. Allograft MNP were phagocytic for latex particles but only very low numbers were adherent to a variety of surfaces. About 50% were positive for attachment and internalization of opsonized sheep red cells via the Fc receptor. However, fewer cells were able to internalize sheep red cells than were able to bind them when complement receptor-mediated phagocytosis was investigated. Large amounts of plasminogen activator were secreted by allograft MNP while cells from syngeneic grafts produced very little. The possible participation of MNP in the effector phase of a mechanism for allograft rejection similar to delayed-type hypersensitivity is discussed.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: I. Ultrastructural and cytochemical features

Mononuclear phagocytes (MNP) have been identified in rejecting rat cardiac allografts by morphological and cytochemical criteria. Their accumulation has been quantitated and their distribution within the graft recorded. Lymphocytes were the major infiltrating cell type present 3 days after transplantation, but by Day 5 and Day 7 there were 2.5 to 3 times as many MNP as lymphocytes. In the later stages (Days 6 and 7) many MNP were closely adjacent to myocardial cells and frequently possessed pseudopodia which were indenting the myocardial cell membrane. Allograft recipients given 750 rads γ-irradiation and reconstituted with thoracic duct lymphocytes rapidly rejected the graft. As many MNP were present in such grafts as in unmodified recipients. A potent antimacrophage serum did not prolong graft survival or alter the numbers of MNP within rejecting grafts. We conclude that MNP must be considered strong candidates for effector cells in allograft rejection and that satisfactory depletion techniques for MNP are not yet available.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: III. The effect of cells extracted from rat cardiac allografts upon beating heart cell cultures

Cells extracted from rat cardiac allografts were able to bring about cessation of beating of heart cell culture monolayers nonspecifically. Nonadherent populations, depleted of macrophages, were consistently less potent than unseparated cells in this assay. Cells extracted from isografts were totally ineffective. Allogeneically stimulated peritoneal cells were also nonspecifically active. Again, nonadherent cells were less efficient than unseparated cells at stopping heart cell monolayers from beating, while adherent cells, enriched for macrophages, were more efficient. Activated bone marrow culture macrophages syngeneic or allogeneic to the heart cultures were also highly potent in beating heart cell assays. Thus in all cases the predominant effector cell type was adherent and nonspecific in its action and therefore presumably a macrophage. Supernatants from wells in which no beating cells remained following incubation with each type of effector population tested were transferred undiluted to fresh wells. In all cases there was no effect at all upon the beating of heart cell monolayers. Antirat heart antiserum plus complement was able to bring about the cessation of beating of heart culture monolayers at a dilution of 1:64. Alloantibody plus complement did not bring about cessation of beating at any dilution, although nonmyocardial cells were killed. The possibility that macrophages are the chief effector cell type in a DTH-like mechanism for cardiac allograft rejection is discussed.     

The coenzyme A requirement of mammalian fatty acid synthetase: evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem.255, 1388–1392). When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl-S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C₆-C₁₆; addition of CoA promoted elongation of the acyl-S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl-N-acetylcysteamine and S-crotonyl-N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl-N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl-S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl-S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa₃ and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c₁, and aa₃, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.     

Biochemical properties of short- and long-chain rat liver microsomal trans-2-enoyl coenzyme A reductase

This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.     

Development of the rat salivary glands. II. The identification of a trypsinlike protease in the submaxillary gland

Trypsinlike protease activity at pH 9.2 was measured in tissue extracts of adult rat salivary glands by using a fluorometric assay in which β-naphthylamine is released by the hydrolysis of benzylarginine β-naphthylamide. The submaxillary gland contains high levels of this activity, and the parotid and sublingual glands have a maximum of 2000-fold and 200-fold less. After polyacrylamide disc gel electrophoresis at pH 8.3, the protease activity of submaxillary extracts is associated with a major protein band. Neither this protein band nor its protease activity is detectable in extracts of parotid or sublingual glands. Homogenates of newborn submaxillary gland do not have this protease activity at detectable levels, suggesting that its major accumulation is postnatal.     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.     

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: Isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2–3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D₃. They metabolize 25-hydroxyvitamin D₃ to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D₃ and the other as its cis isomer. These two “metabolites” have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D₃ on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.     

The preservation of the ability of cultured quail wing bud mesoderm to elicit a position-related differentiative response

A previous study showed that grafting wedges of fresh anterior quail wing mesoderm into posterior slits of chick wing buds resulted in the formation of rods and nodules of cartilage in a high percentage of cases (B. Carlson, 1983, Dev. Biol. 101, 97-105). The purpose of the present study was to determine if a similar response could be elicited by grafting pieces of mesoderm that had been cultured in vitro. When pieces of 1-day cultured anterior mesoderm from stage 17-24 donors were grafted into standard posterior slits of chick wing buds, the percentages of supernumerary structures differed little from those which formed after the grafting of pieces of fresh mesoderm. In a time series, grafts of stage 22-23 anterior mesoderm which had been cultured for 1-4 days retained the ability to form cartilage after being grafted into posterior locations. A time series showed that the duration of this retention was longer in cultured mesoderm than it was in mesoderm that remains in the donor wing bud.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Proteolysis of human reticulocyte membrane proteins: Evidence for a physiological role of the acid endopeptidases

Endogenous membrane proteins, labeled by incubating human reticulocytes with l-[¹⁴C]leucine, are degraded at pH 7.3 by membrane-bound acid proteinases. Solubilized membrane proteins are also degraded at neutral pH by the purified membrane acid proteinases. Exogenous proteins are not degraded by intact membranes and therefore association with the membrane seems to be an essential requirement for protein degradation in the physiological pH range. These findings provide evidence for a physiological function of the enzymes previously characterized as acid proteinases.     

Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney

Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-Sepharose in the presence of Ca2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 mM EGTA. In the presence of free Ca2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein-1 min-1. Maximum stimulation occurred at 0.035-0.076 mM Ca2+. The apparent Ka for calmodulin was 8 nM. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5'-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Tissue fractionation in rat brain, kidney and liver. I. Intracellular localization of a 5-methyltetrahydrofolic acid requiring enzyme.

The intracellular distribution of N-methyl-transferase requiring 5-methyl-tetrahydrofolic acid (5 MT-NMT) was studied in brain, kidney and liver of rats. Among these different tissues, the kidney displayed the highest enzyme activity, more than 20 times the activity detected in the brain. As the striatum and, to a lesser extent the hypothalamus, were found to contain slightly higher 5 MT-NMT than other cerebral regions, they were also selected for the study of the subcellular localization. Tissue fractionation was performed by differential centrifugation yielding five different fractions which were analyzed for their enzymatic content not only of 5 MT-NMT but also of marker enzymes, such as cytochrome oxidase, acid phosphatase and inosine diphosphatase. In all the tissues studied, 5 MT-NMT was recovered in the supernatant fraction. Therefore one may consider this enzyme to belong to the cytosol. Although a neuronal localization cannot be excluded, it is beyond doubt that the enzyme is contained in other cellular types. In the brain fractionation, the five fraction procedure seems to be very useful especially when the subcellular distribution of a given enzyme is compared to that obtained in other tissues like liver or kidney. Finally 5 MT-NMT may be considered a good marker enzyme for the supernatant fraction.     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.