Sudhir K. Mukerji

Corn leaf phosphoenolpyruvate carboxylases: Purification and properties of two isoenzymes

Abstract Two phosphoenolpyruvate carboxylase proteins (PC-I and PC-II) were extracted and purified close to homogeneity from corn leaves. PC-I contained about 85% and PC-II about 15% of the total phosphoenolpyruvate carboxylase activity. PC-I eluted from a DEAE-cellulose column with a buffer having lower ionic strength, had higher K m and V values with respect to phosphoenolpyruvate, Mg 2+ , and Mn 2+ , was more thermolabile and moved more slowly toward the anode during disc gel electrophoresis as compared to PC-II. The enzymes had sedimentation coefficient values ( s 20,W ) of 9.7 and 11.6S and molecular weights, determined by equilibrium centrifugation on sucrose density gradients, of 225,650 and 270,800, respectively. The enzymes used HCO 3 − as the active “CO 2 ” substrate, and the major protein (PC-I) had a temperature optimum for activity of 40 °C.

Corn leaf phosphoenolpyruvate carboxylases. The effect of divalent cations on activity.

Abstract Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg 2+ or Mn 2+ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg 2+ and Mn 2+ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg 2+ -activated system was dependent on a PEP-Mg 2+ complex and not on the concentration of free Mg 2+ or free PEP. Kinetics in the presence of total Mg 2+ and those of PEP-Mg 2+ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction.

Free radical mechanism of oxidation of uroporphyrinogen in the presence of ferrous iron

Human porphyria cutanea tarda is an unusual consequence of common hepatic disorders such as alcoholic liver disease. Hepatic iron plays a key role in the expression of the metabolic lesions, i.e., defective hepatic decarboxylation of porphyrinogens, catalyzed by uroporphyrinogen decarboxylase. This prompted the present study to determine the in vitro effects of iron on the uroporphyrinogen substrate in the absence and presence of atmospheric oxygen. We observed that (i) unless oxygen is the limiting reactant, autoxidation of ferrous iron and iron-catalyzed oxidation of uroporphyrinogen occurred soon after initiating the reaction at pH 7.4 and 30 degrees C in buffers which are non- or poor chelators of iron; (ii) the rates of uroporphyrinogen oxidation were proportional to the initial concentration of ferrous ion; (iii) about 70% of the oxidations of uroporphyrinogen were accountable due to a free-radical chain reaction pathway involving superoxide radical and hence inhibitable by superoxide dismutase; (iv) uroporphyrinogen could be further oxidized to completion by the hydroxyl radical since the reaction was partially inhibited by both mannitol and catalase which prevent hydroxyl radical production; (v) the oxidizing effects of ferric ion on uroporphyrinogen were none or negligible as compared to those of ferrous ion. Ferric was reduced to ferrous ion in the presence of dithiothreitol. When the ferrous ion thus formed was reoxidized in the presence of atmospheric oxygen, minor but definite oxidations of both uroporphyrinogen and dithiothreitol were observed. The oxidations of Fe2+ and uroporphyrinogen could be blocked by 1,10-phenanthroline, a ferrous iron chelator. The data suggest that ferrous is the reactive form of iron that may contribute to pathogenic development of the disease by irreversibly oxidizing the porphyrinogen substrates to nonmetabolizable porphyrins, which accumulate in porphyric liver.

The molecular transformation of rabbit testis proacrosin into acrosin

Abstract Partially purified rabbit testis proacrosin formed only one acrosin of 73,000 ± 3000 apparent molecular weight ( M r ) during the early phase of “autoactivation” at pH 8. Complete “autoactivation” then converted this acrosin to a 38,000 ± 3000 M r , acrosin. These results suggest the existence of a proacrosin dimer (73,000) or a dimer of proacrosin and an acrosomal membrane protein which were converted first to an acrosin dimer (73,000 M r ) or to an acrosin-membrane protein dimer and then to the acrosin monomer (38,000). The formation of a 73,000 ± 3000 M r acrosin from a 73,000 ± 3000 proacrosin is explainable by assuming that either a small activation pertide(s) is released or none at all.

A potential biochemical explanation for the genesis of porphyria cutanea tarda: Studies on the inherent biochemical defect in highly purified human erythrocyte uroporphyrinogen decarboxylase and its amplification by iron

Familial porphyria cutanea tarda (PCT) is a photocutaneous disease in which subnormal activity of uroporphyrinogen decarboxylase is observed both in the liver and red cells. Hepatic iron plays a key role in the genesis of overt biochemical and clinical PCT. In this report, we have studied the properties of 10000‐fold purified erythrocyte uroporphyrinogen decarboxylase preparations from two familial PCT patients and a non‐porphyric control subject. The apparent Michaelis constants (K m), determined by using uroporphyrinogen III substrate, were approx. 3.2‐times higher for the enzyme from the diseased subjects (K m = ~1.0 μM) as compared to the normal (K m = 0.3 μM). Though both abnormal and normal enzymes were inhibited progressively with increasing concentrations of iron, the enzymes from diseased subjects exhibited greater susceptibility e.g. 0.1 mM Fe2+ inhibited the former about 50% and the latter about 20%. These observations suggest that (i) the inherent biochemical defect in PCT is the reduced enzyme‐substrate affinity and (ii) the intrinsic abnormal conformation renders the PCT enzyme particularly susceptible to inhibition by iron.

Conversion of rabbit testis proacrosin to acrosin

The mammalian sperm trypsin-like enzyme acrosin (E.C. 3.4.21 .lO) appears to play an important role in fertilization [ 1 ] . Work from this laboratory on rabbit testis has suggested that rabbit testis acrosln is present as a zymogen, proacrosin [2,3], and our preliminary studies have also indicated this proacrosin is present in rabbit epididymal sperm [4]. The aim of the present communication is to present new evidence which supports the zymogen nature of rabbit testis proacrosin, and evidence which strongly suggests that proacrosin can be activated by an autocatalytic process regulatable by Zr?’ and Ca2’.

Corn leaf phosphoenolpyruvate carboxylases: Inhibition of 14CO2 fixation by SO32− and activation by glucose 6-phosphate☆☆☆

Abstract Sulfite ion, the hydrated form of SO 2 which is an air pollutant, was found to be an inhibitor of phosphoenolpyruvate carboxylase(s) isolated from corn leaves. The inhibition was partial even in the presence of excess SO 3 2− . It inhibited the enzyme competitively with respect to HCO 3 − , noncompetitively with respect to phosphoenolpyruvate, and uncompetitively with respect to Mg 2+ . The kinetics of inhibition suggest that an alternate pathway is operative in the presence of SO 3 2− . The enzyme(s) were activated by glucose 6-phosphate which affected primarily the affinity of the enzyme for phosphoenolpyruvate. The binding site of glucose 6-phosphate was apparently distinct from the catalytic site of the enzyme since partial destruction of the catalytic site by heat had no effect on the inhibition by SO 3 2− , but glucose 6-phosphate lost its activating effect. The inhibition due to SO 3 2− was relieved by glucose 6-phosphate.

Uroporphyrinogen decarboxylases from human erythrocytes : purification, complete separation and partial characterization of two isoenzymes

1. Two distinct molecular forms of uroporphyrinogen decarboxylase have been completely separated and highly purified from human erythrocytes. 2. Each protein, with molecular masses of about 52-54 kDa and 35 kDa, are apparently composed of a single polypeptide chain. 3. They may form a functional decarboxylating complex for heme biosynthesis.