Low Molecular Weight Enzyme Research Articles

An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves

Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm(3).High molecular weight (280,000) malate dehydrogenase could be converted to active low molecular weight (69,000) malate dehydrogenase by treatment with 2.5 m CsCl, 1.0 m NaCl, 6 m urea, pH 6.5 or below, or one freeze-thaw cycle. Simple removal of salt or raising the pH were not effective in reforming the high molecular weight malate dehydrogenase after dissociation. The high molecular weight enzyme was not dissociated during prolonged dialysis against 0.1 m NaCl or 0.05 m phosphate, pH 7.0. Calcium at concentrations up to 0.1 m produced no activation or differential response in the two MDH forms.High and low molecular weight malate dehydrogenase were nearly identical in susceptibility to inhibition by various unreactive substrate analogs. However, there was a marked difference in the ability of the two forms of malate dehydrogenase to reduce 3-acetylpyridine-deamino-NAD. This difference in activity was the basis of a convenient assay for determining the ratio of high to low molecular weight malate dehydrogenase in crude extracts. The pH activity profiles and Michaelis constant for malate were nearly identical for the two molecular weight forms.Analysis by polyacrylamide gel electrophoresis revealed one high molecular weight and two low molecular weight malate dehydrogenase zones. Dissociation of high molecular weight malate dehydrogenase resulted in formation of low molecular weight enzyme whose electrophoretic properties differed from the normal low molecular weight forms.

Human N-acetyl-β-hexosaminidases: Hydrolysis of N, N′diacetylchitobiose by a low molecular weight enzyme

Human N-acetyl-β-hexosaminidases: Hydrolysis of N, N′diacetylchitobiose by a low molecular weight enzyme

Enzymatically active intermediate in the conversion between the low and high molecular weight DNA polymerase.

MOST eukaryotic cells contain at least two types of DNA polymerase—a low molecular weight (3.5S) enzyme found predominantly in the nucleus and a heterogenous high molecular weight (6–8S) enzyme(s) isolated from the cytoplasmic fraction1–4. Although the physiological relationship between these activities is not yet understood, recent studies have shown that it is the cytoplasmic polymerase that responds to the stimulus for cellular proliferation5,6. The role this enzyme could play in DNA replication in vivo is not certain; most DNA is located in the nucleus and the nuclear (3.5S) enzyme can catalyse a single round of complementary synthesis in vitro1. One way to resolve this apparent paradox is to propose a precursor relationship between the cytoplasmic and nuclear DNA polymerases. Such a model is supported by evidence for the existence of a common component in the two enzymes. Chang and Bollum8, using rabbit antibody prepared against a high molecular weight DNA polymerase, have found common antigenic determinants existing in both activities. We have shown that an active 3.5S component can be dissociated from the high molecular weight activity by the addition of salt. This subunit appears identical to the nuclear 3.5S enzyme by several enzymatic and physical criteria9,10. Furthermore, this subunit can be reconverted reversibly to a high molecular weight DNA polymerase by the removal of the salt4,9. I present here evidence for the existence of an intermediate in the in vitro conversion between the low and high molecular weight DNA polymerases.

Studies on 5′-nucleotidases of rat liver

Approximately 8% of the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity (assayed with AMP as substrate at a pH of 7.4 in the presence of 10 mM tartrate) in a homogenate of rat liver behaved as a lysosomal enzyme when the homogenate was fractionated into its subcellular components by differential and density gradient centrifugation. The bulk of the activity sedimented with the nuclear and microsomal fractions. The 5′-nucleotidases in purified lysosomal and plasma membrane fractions exhibited nearly maximal activity at pH values between 7.0 and 9.5, and were similarly influenced by the addition of varying concentrations of Mg 2+ and Mn 2+ to the incubation mixture. When a lysosomal fraction solubilized by treatment with 1% Triton X-100 was passed through a Sephadex G-200 column, two peaks possessing 5′-nucleotidase activity appeared in the effluent. The first peak was identical chromatographically to the 5′-nucleotidase associated with plasma membranes and with a lysosomal membrane fraction. The second peak exhibiting 5′-nucleotidase activity contained a lower molecular weight enzyme which could be extracted from lysosomes by 1 mM NaHCO 3. Disc electrophoresis of these same fractions on polyacrylamide gel confirmed these chromatographic findings.

ATP:Glutamine synthetase adenylyltransferase from Escherichia coli: Purification and properties of a low-molecular weight enzyme form

Some kinetic properties of a low-molecular weight form of the ATP: glutamine synthetase adenylyltransferase of Escherichia coli are reported. Described also is a purification procedure from which the small adenylyltransferase of ~70,000 molecular weight is purified ~500-fold from E. coli extracts and is derived from a larger active protein (~130,000 molecular weight) that is a component of the deadenylylating enzyme system of this microorganism. The small adenylyltransferase isolated can be purified a further 1.7-fold in polyacrylamide gel electrophoresis. The incorporation of 5′-adenylyl groups into the glutamine synthetase structure is optimal at pH ≥ 7.8, 30 m m MgCl 2 (15 m m MnCl 2 is about one-third as effective an activator as is MgCl 2), 10 m m l-glutamine, and 5 m m ATP. Anions, α-ketoglutarate, UTP, l-glutamate, various divalent cations with MgCl 2 or excess MgCl 2 (MnCl 2), and sulfhydryl reagents are inhibitory. The adenylyltransferase and unadenylylated glutamine synthetase have an apparent association constant of ~5 × 10 5 m −1. With pyrophosphate present, the small adenylyltransferase catalyzes the removal of 5′-adenylyl groups from adenylylated glutamine synthetase, and ATP has some activating effect on this reverse reaction. Dodecameric glutamine synthetase that has been coupled to Sepharose 4B is enzymatically active and has been used as a solidstate substrate for some experiments with the adenylyltransferase. The adenylyltransferase, however, is inactive with subunits of glutamine synthetase that have been reacted with p-chloromercuriphenylsulfonate and attached to Sepharose 4B to prevent their reassociation during the removal of denaturants.

Lysozyme excretion as a measure of renal tubular dysfunction in children.

1. Activity of the low molecular weight enzyme lysozyme was measured in the plasma and urine of healthy adults and children and of children with renal disease. 2. No difference was detected between lysozyme excretion (expressed as the lysozyme/creatinine clearance ratio) of healthy adults and neonates, implying that the proximal tubular function of protein reabsorption is mature in the neonate. 3. The lysozyme/creatinine clearance ratio was not elevated in the nephrotic syndrome: thus a heavy load of filtered albumin does not interfere with low molecular weight protein reabsorption. 4. Very high values of lysozyme/creatinine clearance were observed in children with the Fanconi syndrome; there was no overlap with any other group studied. 5. Children with pyuria had a very slight increase in urine lysozyme/creatinine concentration ratio.

On the fragmentation of mitochondria by diethylstilbesterol: II. On the relation of the released proteins to the mitochondrial membranes

The fragmentation of mitochondria by the combined action of diethylstilbesterol andMg ++ can be controlled by the level of added Mg ++. At concentrations of 0.01 m or less of Mg ++, some 20% of the total protein is solubilized and the low molecular weight enzymes of the citric acid cycle are quantitatively recovered. However, the high molecular weight enzymes of the citric acid cycle are released only in small yield. At concentrations of Mg ++ approaching 0.1 m, some 30% of the total protein is released and good recoveries of the enzymes of the citric acid cycle are observed, whether these are of the low or high molecular weight category. It can be shown that under conditions of protein release, a significant amount of phospholipid is also released. This released phospholipid cosediments with the membranous residue but is separable in crude form from the cristael vesicles by gradient centrifugation. Electron microscopic studies have shown that under optimal conditions of fragmentation by diethylstilbesterol and Mg ++, the boundary membranes of the mitochondrion completely disappear. The membranous residue has been identified with the cristael membranes on the basis of enzymic activity, composition and electron microscopy. The soluble fraction contains the same set of enzymes which has previously been assigned to the boundary membranes on the basis of other fragmenting procedures. It can be shown that authentic inner membrane particles do not release soluble protein when exposed to the combined action of diethylstilbesterol and Mg ++. The available evidence is compatible with the thesis that the primary event in the fragmentation of mitochondria by diethylstilbesterol plus Mg ++ is the dissolution of the boundary membranes with concomitant solubilization of mitochondrial protein and release of a phospholipid-enriched fraction.

Properties and subcellular distribution of ornithine cycle enzymes in amphibian kidney

1. 1. The kidneys from amphibian species which transport urea either actively ( Rama catesbiana) or passively ( Bufo paracnemis, Bufo. arenarum, Phylomedusa sauvagyi and Ceratophis ornata) have high and low arginase concentrations, respectively. 2. 2. The latter species have other ornithine cycle enzymes physically located in a fashion compatible with urea synthesis. 3. 3. Amphibian renal arginase, whether capable or not of hydrolysing endogenous arginine, is similar to the low molecular weight enzyme of the liver of ureotelic animals. 4. 4. Bullfrog renal arginase is not a structural component of the membrane of the tubular cells.

Solubilization and Comparative Properties of Some Mammalian Diphosphopyridine Nucleosidases

Abstract Diphosphopyridine nucleosidases (DPNases) were solubilized from acetone powders prepared from some mammalian tissues. Solubilization was effected by the action of porcine pancreatic lipase on DPNases that are resistant to solubilization by trypsin and which heretofore had not been solubilized. Molecular weight estimates made on DPNases obtained from porcine, bovine, sheep, dog, horse, mouse, rabbit, and human tissues, as well as mouse Ehrlich ascites tumor cells indicate that these enzymes fall into three size classes: porcine, bovine, and sheep, ∼25,000; dog, ∼50,000; and the remainder between 70,000 and 90,000. The electrophoretic characteristics of the DPNases indicate that with the exception of the sheep brain material all the low molecular weight enzymes migrate toward the cathode at pH 7.0. The sheep DPNase as well as the enzymes from all the other sources studied migrate in the opposite direction. The mouse ascites cell, rat, rabbit, and sheep preparations are particularly interesting in that they show a number of multiple forms that are enzymatically active. Notwithstanding the variability in molecular weight and electrophoretic mobility, the solubilized mammalian DPNases exhibit similar although not identical catalytic properties in their sensitivity to nicotinamide inhibition, specificity to DPN+ and TPN+, and formation of the 3-acetylpyridine analogue of DPN+. A new catalytic stain is described that is specific for DPNases which carry out the exchange reaction with pyridine bases.

Reversible inhibition of pepsin by polylysine.

THE mode of action of low-molecular weight enzyme inhibitors has been intensively studied. This does not, however, apply to high-molecular weight enzyme inhibitors. So far as we are aware, no work has been published on synthetic high-molecular weight enzyme inhibitors, and the mode of action of natural high-molecular weight enzyme inhibitors, such as the pepsin inhibitor and the various trypsin inhibitors, is still not fully understood.