Inactive Form Of Enzyme Research Articles

Autophosphorylation of Enzyme I of theEscherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Requires Dimerization

Enzyme I of theEscherichia coliphosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition.In vitroautophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity blotting assay for 2-5A-dependent RNase

The intracellular effectors known as 2-5A (ppp(A2′p) n A) regulate the cleavage of single-stranded RNA by activating a latent endoribonuclease (2-5A-dependent RNase or RNase L). Accordingly this enzyme may exist in either an inactive form, free of 2-5A, or an active, 2-5A-bound form. Previously, a radiobinding assay for 2-5A-dependent RNase was developed that measured the amount of labeled ppp(A2′p) 2A3′-[ 32P]Cp, a derivative of ppp(A2′p) n A, that bound the inactive enzyme form. Because 2-5A-dependent RNase has a particularly high affinity for 2-5A the radiobinding assay may not measure the 2-5A activated form of the enzyme. Therefore an efficient procedure to facilitate the detection of total 2-5A-dependent RNase (i.e., 2-5A-free and 2-5A-bound enzyme) in mouse spleen extracts was developed. Denaturing conditions were used to ensure that all 2-5A-dependent RNase was in the 2-5A-free form. After denaturation on polyacrylamide gel electrophoresis, optimal blotting conditions onto nitrocellulose and renaturation of the 2-5A binding site of 2-5A-dependent RNase were developed. This procedure allowed a population of enzyme that otherwise is not accessible by the classical radiobinding assay to be assayed, thus leading to an increased measurement of 15–17% in cytoplasmic spleen extracts.

Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.

Crystal structure of unliganded phosphofructokinase from Escherichia coli

In an attempt to characterize the mechanism of co-operativity in the allosteric enzyme phosphofructokinase from Escherichia coli, crystals were grown in the absence of activating ligands. The crystal structure was determined to a resolution of 2.4 Å by the method of molecular replacement, using the known structure of the liganded active state as a starting model, and has been refined to a crystallographic R-factor of 0.168 for all data. Although the crystallization solution would be expected to contain the enzyme in its inactive conformation, with a low affinity for the co-operative substrate fructose 6-phosphate, the structure in these crystals does not show the change in quaternary structure seen in the inactive form of the Bacillus stearothermophilus enzyme (previously determined at low resolution), nor does it show any substantial change in the fructose 6-phosphate site from the structure seen in the liganded form. Compared to the liganded form, there are considerable changes around the allosteric effector site, including the disordering of the last 19 residues of the chain. It seems likely that the observed conformation corresponds an active unliganded form, in which the absence of ligand in the effector site induces structural changes that spread through much of the subunit, but cause only minor changes in the active site. It is not clear why the crystals should contain the enzyme in a high-affinity conformation, which presumably represents only a small fraction of the molecules in the crystallizing solution. However, this structure does identify the conformational changes involved in binding of the allosteric effectors.

Glutathione S-transferase 1 and 2 in susceptible and insecticide resistant Aedes aegypti

Glutathione S-transferase enzymes (GST) were characterized in permethrin and in DDT resistant and susceptible strains of Aedes aegypti. The predominant GST, designated GST-1, was purified from susceptible and resistant larvae using S-hexyl-glutathione affinity chromatography and anion-exchange chromatography. Rabbit antiserum directed against the 26.8-kDa GST-1 subunit purified from the susceptible strain was specific for a 26.8-kDa subunit in the resistant strain. There was no significant difference in enzyme kinetic constants ( K m and V max) between GST-1 from susceptible and resistant strains. GST-1 enzyme activity varied during larval development in both resistant and susceptible strains, but was approximately 1.7 times greater in resistant larvae and pupae throughout development. The GST-1 amount (measured using a radioimmunoassay) also varied during development, and was generally higher in resistant larvae and pupae. GST-1 enzyme activity measured each day during the 6 days of larval development was significantly correlated with GST-1 protein amount on each of the previous days, suggesting that an inactive enzyme form is converted to the active form approximately 24 hr after it is synthesized. Another GST enzyme, GST-2, was also found in resistant and susceptible A. aegypti larvea. GST-2 was partially purified from resistant larvae using S-2,4-dinitrophenylglutathione affinity chromatography and anion-exchange chromatography. GST-2 has a molecular mass of 28 kDa, and an isoelectric point of less than 5.0. GST-2 was not cross-reactive with GST-1 antiserum. GST-2 enzyme activity varied during the development of both susceptible and resistant larvae, but the pattern was different than that for GST-1. Throughout development, GST-2 enzyme activity was substantially higher (approximately eight-fold) in resistant strains of A. aegypti compared to susceptible strains.

Cross-reactivity of polyclonal and monoclonal antibodies to polyphenoloxidase in higher plants

Polyclonal and monoclonal antibodies against broad bean polyphenoloxidase were used to examine the cross reactivity of the enzyme in higher plants after Western blotting. Both the polyclonal and monoclonal antibodies recognized similar enzyme forms in broad beans and mung beans, but only the polyclonal antibody identified polyphenoloxidase in pea and soybean extracts. The polyclonal antibodies were able to identify inactive and/or active enzyme forms of polyphenoloxidase in the various plant sources under partially denaturing SDS-PAGE. The polyclonal antibody was also used to identify several cross-reacting proteins in five other plant species after denaturing SDS-PAGE. Under denaturing SDS-PAGE, the number and types of polyphenoloxidase identified were surprisingly similar among the eight species and were localized to proteins with M r s of 60–63 000, 43–45 000, and 34–36 000.

Correlation between hysteresis and allosteric properties for phosphofructokinase from Ascaris suum.

The Ascaris suum phosphofructokinase exhibits hysteretic transitions in the time course for fructose 6-phosphate (F6P) phosphorylation in addition to allosteric properties when assayed at pH values below 8. Conditions that enhance hysteretic changes also enhance cooperative interactions and thus there appears to be a link between hysteresis and cooperativity. Initiation of reaction with either F6P or phosphofructokinase results in a pronounced lag, while initiation of the reaction with MgATP results in a burst at pH values below 8. Under conditions in which a lag is evident, increasing the concentration of F6P in the assay decreases the lag, while under conditions where a burst is evident, increasing the concentration of MgATP in the assay decreases the burst. The lag is enzyme-dependent going to a limiting value at high enzyme concentration, while the burst is enzyme-independent. As the pH increases, the Hill coefficient for F6P decreases from a pH-independent value of 3 at low pH to a value of 1 above pH 8. Over the same pH range, the burst rate increases to a point that it is too fast to measure at pH 8 (that is, the time course is linear). Finally, at pH 6.9, the saturation curve for F6P becomes more cooperative with the Hill coefficient equal to 3 above 4 mM MgATP. Data are interpreted in terms of the model suggested for the rabbit skeletal muscle phosphofructokinase (Frieden, C., Gilbert, H. R., and Bock, P.E. (1976) J. Biol. Chem. 251, 5644-5647) in which MgATP binds preferably to an inactive tetrameric enzyme form in which a group with a pK of 6.8 is protonated and F6P binds preferably to the unprotonated active tetrameric form.

Active, inactive and in vitro synthesized forms of polyphenoloxidase during leaf development

Polyphenoloxidase (PPO; EC 1.14.18.1) activity decreased 8‐fold from young to mature Vicia faba L. Moensch (cv. Long pod) leaves. The Km for catechol remained relatively constant from young to mature leaves. Electrophoretic separation and analysis showed that only one active form was present in extracts from various leaf sizes. The amount of this form appeared to decrease with leaf size/age. In extracts which had not tanned, electroblotting and immunostaining indicated that one enzyme form was present with a molecular mass of 45 kDa. Two leaf categories contained greater amounts of this immunological cross‐reacting PPO than other leaf categories. When extracts were allowed to darken, immunoblotting detected three enzyme forms with molecular masses of 45, 59 and 63 kDa. The latter two immunological crossracting species had no detectable enzyme activity. Poly‐A+ mRNA was isolated from six leaf sizes and translated in vitro. A product corresponding to PPO was present in all leaf categories. Greater amounts of this translation product were observed in medium‐sized leaves than in very young or mature leaves. These results suggest that: (1) enzymatic assays for PPO are not reliable indicators of the total amount of PPO protein present in developing leaves, (2) immunoblotting can detect inactive enzyme forms, (3) only one active form of the enzyme is present at all developmental stages, and (4) mRNA corresponding to PPO is present at all developmental stages but appears to be more abundant in certain leaf sizes/ages.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: Chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 °C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 m m dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.

Relationship Between Steady-State Gas Exchange, in vivo Ribulose Bisphosphate Carboxylase Activity and Some Carbon Reduction Cycle Intermediates in Raphanus sativus

The relationships between CO2 assimilation rate, RuP2 carboxylase activity and sizes of the pools of ribulose 1,5-bisphosphate (RuP2) and 3-phosphoglyceric acid (PGA) were examined using a freeze clamp device to rapidly freeze sections of attached leaves of R. sativus which previously had gas-exchange measurements made on them. At high irradiance and ambient partial pressures of CO2 and O2, RuP2 carboxylase was fully active in vivo. Activity was less at very low CO2 pressures and at high CO2 pressures, particularly when combined with low O2 pressures. In vivo RuP2 carboxylase activity and both RuP2 and PGA pool sizes increased with increasing irradiance. RuP2 pool sizes were high at low CO2 pressures and decreased at high CO2 pressures. PGA pool sizes, on the other hand, were low at low CO2 and high at high CO2 pressures. A model of RuP2 carboxylase-oxygenase (Rubisco) kinetics is used to examine the quantitative relationship between in vivo RuP2 carboxylase activity, CO2 assimilation rate and RuP2 and PGA pools. The model predictions fit in vivo data, except at high CO2 pressures, if it is assumed that RuP2 does not bind tightly to the inactive enzyme form in vivo. It is shown that a large fraction of the RuP2 and PGA pools may be chelated by magnesium in the stroma and that the high RuP2 pools (e.g. at low irradiance) may represent an optimal concentration rather than be truly saturating. We conclude that RuP2 pool sizes above Rubisco site concentration do not necessarily indicate a Rubisco limitation of photosynthetic rate.

Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components

Monoclonal antibodies that react with antigens of the plasma membrane of rat intestinal villus and crypt cells have been prepared by fusion of mouse myeloma (NSI) cells with spleen cells of mice immunized with various intestinal cellular fractions, including the luminal membrane of adult villus and crypt cells, and of newborn rat intestinal cells. The antigenic targets of most antibodies have been identified. They include major protein components of the brush border (luminal) membrane of adult villus cells (sucrase-isomaltase, maltase, lactase, aminopeptidase N, alkaline phosphatase) and newly identified protein antigens specific for intestinal epithelial cells. Of 25 independently derived monoclonal antibodies prepared, 18 reacted exclusively with the brush border membrane of the villus cells, confirming its unique protein composition. Antibodies specifically staining the crypt cells, the newly differentiated epithelial cells present in the lower half of the villi, the top villus cells, and both villus and crypt cells were also obtained and characterized. These antibodies have been used to study the expression of cell- and tissue-specific functions during differentiation and development of the intestinal epithelium. Contrary to results obtained with polyclonal antisera, no inactive forms of the brush border enzymes have been detected in the crypt cells. The identification of cell surface components expressed at different levels of the villi, and in both undifferentiated and differentiated intestinal cells, suggests that cell differentiation in the intestinal epithelium is a continuous and gradual process involving both transcriptional and translational regulation of different sets of genes.

Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa.

Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.

Demonstration of renin activity in purified rat Leydig cells: evidence for the existence of an endogenous inactive (latent) form of enzyme.

Previous histochemical studies demonstrated the specific localization of immunoreactive renin-like substance in Leydig cells of rat testes. The studies reported herein demonstrate a specific renin enzyme activity in the two purified populations of Leydig cells (I and II) of mature rat testes. Leydig cells of both populations also exhibited an inactive (latent) renin which was activated by the sulfhydryl reagents dithiothreitol, beta-mercaptoethanol, glutathione, and cysteine but not by limited proteolysis by trypsin, which is a characteristic activating agent for prorenin or inactive renin of the zymogen type. The activation of latent renin by dithiothreitol produced approximately 5-and 10-fold increases in renin activity in Leydig cell populations I and II, respectively. Active and latent renin showed strong affinity to an antirat renin immunoglobulin-Sepharose column, indicating a close immunological relationship of latent renin to active renin. Both active and latent renin from Leydig cell populations (I and II) exhibited the pH optimum of 6.0. The gel filtration of Leydig cell extracts characteristically revealed that the apparent mol wts of active and latent renin were 39,000 and 48,000, respectively. Both active and latent renin in Leydig cells remained almost at similar levels through four continuous subcultures. The activity of latent renin slightly increased during the four consecutive subcultures, while active renin levels remained almost constant.

Steady-state kinetics of horse-liver alcohol dehydrogenase with a covalently bound coenzyme analogue.

The steady-state kinetics of the enzyme modified by affinity labelling with NAD analogue, nicotinamide-N6-[N-(6-aminohexyl)carbamoylmethyl]-adenine dinucleotide, has been investigated using a recycling reaction with p-nitrosodimethylaniline and n-butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping-pong mechanism involving two inactive enzyme forms (enzyme-NAD and enzyme-NADH complexes in the 'open' conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p-nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive 'open' form of the enzyme-NADH complex is negligible, whereas the 'open' form of the enzyme-NAD complex seems to represent a more significant portion (about 30%) under the conditions used.

Inhibition of ribulose bisphosphate carboxylase by substrate ribulose 1,5-bisphosphate.

Substrate ribulose bisphosphate is a potent and a weak inhibitor of the rate of CO2/Mg2+ activation in the carboxylase purified from spinach leaves and Rhodospirillum rubrum, respectively. At 2 degrees C, the concentration of ribulose bisphosphate required for 50% inhibition of the initial rate of CO2/Mg2+ activation was less than 0.4 microM for the spinach enzyme, but between 67 and 270 microM for the R. rubrum carboxylase. Activator 14CO2 trapping experiments demonstrated that ribulose bisphosphate inhibits activation by excluding activator CO2 from the spinach enzyme. The reason for the different sensitivities to inhibition by substrate was evident from equilibrium binding studies with the inactive enzyme forms which indicated that the KD (ribulose bisphosphate) was 0.021 microM for spinach enzyme and 5.9 microM for the R. rubrum protein. Inhibition of activation, however, was not explained by the equilibrium binding results alone. Ribulose bisphosphate was observed to dissociate very slowly from the inactive spinach enzyme (at 2 degrees C, kOFF = 4.9 X 10(-5) s-1). The release of substrate from the inactive R. rubrum carboxylase was much more rapid, with a minimum value for kOFF estimated at 5 X 10(-3) s-1 at 2 degrees C. We conclude that strong inhibition of CO2/Mg2+ activation in the spinach enzyme is mediated by the tight binding and slow release of ribulose bisphosphate, which prevent activator CO2 and Mg2+ from binding to the protein. Weak inhibition of activation in the R. rubrum enzyme results from a larger KD value and a more rapid exchange of ribulose bisphosphate, which allow activator CO2 and Mg2+ to bind to the free enzyme between successive substrate-binding events.

Hepatic Collagenase Activity During Carbon Tetrachloride Induced Fibrosis

Hepatic Collagenase Activity During Carbon Tetrachloride Induced Fibrosis. Lindblad, W.J. and Fuller, G.C. (1983). Fundam. Appl. Toxicol. 3:34-40. The contribution of collagen degradation, as measured by collagenase activity, to the accumulation of collagen during hepatotoxic fibrogenesis was examined using a carbon tetrachloride rat model. Both active and inactive enzyme forms were determined with the inactive form quantitated following activation by limited trypsin digestion of the liver homogenate. The rate of collagen biosynthesis was monitored by quantitating hepatic prolyl hydroxylase activity and accumulation of collagen. In one study CCl4 was administered twice weekly, i.p. (0.2 mL, 33% v/v in light mineral oil) for sixteen weeks, with animals sacrificed every two weeks. Histologic examination of liver sections and serum alanine transaminase levels indicated a progressive necrosis and fibrosis which was confirmed by the increase in hepatic hydroxyproline content from 0.303 to 4.84 μg/mg wet wt. tissue. Prolyl hydroxylase activity increased in a time dependent manner to a maximum of 3.7 × control. This increase was accompanied by a large increase in both active collagenase (15.0–40.8 mU/mg protein) and latent collagenase activity (69.8–191.7 mU/mg protein). These increases were maintained through the transition to irreversible fibrosis. The increase in active collagenase activity was positively correlated with collagen content. A second short term CCl4 treatment study confirmed a transient alteration in the active to latent collagenase ratio early in the fibrotic process. These results demonstrate the dynamic changes in hepatic collagenase levels and indicate that the fibrotic lesion is not dependent on decreased collagenase levels for collagen accumulation.

Hepatic collagenase activity during carbon tetrachloride induced fibrosis

The contribution of collagen degradation, as measured by collagenase activity, to the accumulation of collagen during hepatotoxic fibrogenesis was examined using a carbon tetrachloride rat model. Both active and inactive enzyme forms were determined with the inactive form quantitated following activation by limited trypsin digestion of the liver homogenate. The rate of collagen biosynthesis was monitored by quantitating hepatic prolyl hydroxylase activity and accumulation of collagen. In one study CCl 4 was administered twice weekly, i.p. (0.2 mL, 33% v/v in light mineral oil) for sixteen weeks, with animals sacrificed every two weeks. Histologic examination of liver sections and serum alanine transaminase levels indicated a progressive necrosis and fibrosis which was confirmed by the increase in hepatic hydroxyproline line content from 0.303 to 4.84 μg/mg wet wt. tissue. Prolyl hydroxylase activity increased in a time dependent manner to a maximum of 3.7 × control. This increase was accompanied by a large increase in both active collagenase (15.0–40.8 mU/mg protein) and latent collagenase activity (69.8–191.7 mU/mg protein). These increases were maintained through the transition to irreversible fibrosis. The increase in active collagenase activity was positively correlated with collagen content. A second short term CCl 4 treatment study confirmed a transient alteration in the active to latent collagenase ratio early in the fibrotic process. These results demonstrate the dynamic changes in hepatic collagenase levels and indicate that the fibrotic lesion is not dependent on decreased collagenase levels for collagen accumulation.

Suppressed collagenolytic activity in polymorphonuclear leucocytes from diabetic humans

Extracts of polymorphonuclear leucocytes (PMNL) from diabetic human exhibited less collagenolytic activity than extracts from normoglycemic control subjects. Partially purified control extracts produced αA and αB collagen breakdown products of the type generated by mammalian collagenase; the diabetic preparation produced decreased amounts of the same products. The diabetic PMNLs may synthesize abnormally low levels of collagenase or contain inactive forms of this enzyme.

Radioiodinated antibodies, a tool in studies on the presence and role of inactive enzyme forms: Regulation of chalcone synthase in parsley cell suspension cultures

A new technique, the quantitative determination of total enzyme concentrations by specific immunoprecipitation with purified, radioiodinated antibodies, was used to investigate the presence and possible roles of inactive enzyme in the regulation of chalcone synthase. Dark-grown cell suspension cultures from parsley ( Petroselinum hortense) contained neither catalytically active nor detectable amounts of immunoprecipitable chalcone synthase. Irradiation induced large increases and subsequent decreases of both. Significant differences in the peak positions and in the half-lives of active and total chalcone synthase indicated that induced cells contained inactive as well as active enzyme forms. The presence of inactive enzyme could be explained by two different modes of regulation, (i) simultaneous de novo synthesis of active and inactive enzyme (“Simultaneous Model”), or (ii) de novo synthesis of active enzyme only, with sequential steps of inactivation and degradation (“Sequential Model”). Both models were compatible with experimental results, as analyzed mathematically by investigating the relations between curves for rate of enzyme synthesis, enzyme activity, total enzyme, and half-lives of active and total enzyme. However, the “Simultaneous Model” postulated that de novo synthesis of inactive enzyme represented always the vast majority of total enzyme synthesis, while the Sequential Model integrated inactive enzyme with facility in a sequence of irreversible inactivation and degradation of active enzyme. Experiments with repeated induction indicated that cells containing large amounts of inactive enzyme increased enzyme activity by de novo synthesis rather than by activation of preexisting inactive enzyme.