Kinetic mechanism of guinea-pig skeletal muscle lactate dehydrogenase (M4) with oxaloacetate-NADH and pyruvate-NADH as substrates

• 1.1. Guinea-pig skeletal muscle lactate dehydrogenase M₄ isoenzyme, with pyruvate and NADH as substrates, is adapted to an ordered bi-bi ternary complex mechanism at pH 7.0.
• 2.2. In the same conditions, the kinetic mechanism of the reaction, with oxaloacetate and NADH as substrates, is of the rapid equilibrium ordered bi-bi ternary complex type; NADH is the first substrate in the reaction sequence.

     

Kinetic mechanism of the molecular forms of chicken liver mitochondrial malate dehydrogenase

• 1.1. The reaction kinetic mechanism (pH 7.4) of the molecular forms of chicken liver m-MDH is of the ordered bi-bi ternary complex type with the existence of the E-oxaloacetate, E-L-malate, E-NAD⁺ oxaloacetate, E-NADH-l-malate, E-NAD⁺-NADH, E-NAD⁺-NAD⁺, E-NADH-NAD⁺ and E-NADH-NADH abortive complexes.
• 2.2. The saturating concentration values of the substrates are notably modified, in certain cases, in the presence of the reaction products.

     

The breakdown of Tetrahymena ribosomes by lysosomal enzymes: Inhibition by cytosol

• 1.1. Extracts from Tetrahymena lysosomes contained acid RNase and proteinase. At pH 7.4 there was appreciable proteinase activity which was inhibited by a heat-stable protein present in cell sap.
• 2.2. Lysosomal enzymes rapidly converted 80S ribosomes to subunits at pH 7.4. Hydrolysis of ribosomal RNA was very slow at pH 7.4 but rapid at pH 5.0.
• 3.3. These reactions were inhibited by proteinase inhibitors and by cell sap, but the latter was relatively ineffective at pH 5.0.
• 4.4. It seems unlikely that ribosome breakdown in vivo is initiated by the release of lysosomal enzymes into the cytosol.

     

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum

• 1.1. Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000–400,000 with a peak at about MW 200,000.
• 2.2. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000–600,000 (77% of the fraction NSILA or 28% of total serum NSILA).
• 3.3. Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000–200,000 and 35,000–100,000 fractions and corresponded to 37% of total serum NSILA.
• 4.4. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000–100,000.
• 5.5. Low MW NSILA (<15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000–600,000. This complex was not bound by Con-A Sepharose.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

Effect of caffeine,theophylline and nicotine on d-glucose and folate transport in rat jejunal brush border membrane vesicles

• 1.1. Nicotine at 10 mM, but not caffeine or theophylline, reduced by 20% the overshoot of the Na⁺-dependent d-glucose transport in ratjejunal brush border membrane vesicles.
• 2.2. Since nicotine did not affect the transport of Na⁺, its inhibition on Na⁺-dependent d-glucose transport must be due to a direct effect upon the d-glucose transport system.
• 3.3. Folate transport in these membrane vesicles was found to a be a free diffusion process at pH 7.4.
• 4.4. Neither caffeine, theophylline nor nicotine has any effect on folate transport.

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

The precipitation of human IgG and its subunits with heparin

• 1.1. About 40% of IgG is precipitated when heparin is added to human plasma at pH 5.4, while only a little IgM and no IgA are precipitated.
• 2.2. The precipitation of purified IgG by heparin is pH-dependent and follows a sigmoid curve between pH 7.0 and 5.0.
• 3.3. The precipitate has a constant molar ratio heparin: IgG, independent of pH or the amount of heparin that is added.
• 4.4. The precipitate does not redissolve at high heparin concentrations.
• 5.5. The heavy chains of IgG precipitate also at pH 5.4, but this precipitate redissolves in excess heparin.
• 6.6. Light chains do not precipitate and the F_ab and F_c fragments are only partly precipitated.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Effects of temperature and acid stress on hatching,survival capacity,metabolism and postural reflexes in caenid mayflies (ephemeroptera)

• 1.1. Mortality was 100% at pH 3.5 over a temperature range of 10–30°C for embryos and nymphs of Caenis diminuta and C. hilaris.
• 2.2. Hatching success for both species was highest at pH values above 4.5.
• 3.3. Survival capacities were significantly higher at 20°C over a pH range of 4.0-7.2.
• 4.4. Oxygen consumption rates increase as a function of increasing temperature and reduced acidity.
• 5.5. Loss of the nymphal righting response was observed at pH 3.5. This response can be used as a behavioral assay for acid stress.

     

Breast,hips, and buttocks revisited: Honest fatness for honest fitness

This paper comments on: Low, B. S., Alexander, R. D., and Noonan, K.M. Human hips, breast, and buttocks: Is fat deceptive? Ethology and Sociobiology 8: 249-247, 1987. In it I argue that:

• 1.1. Sexual selection has probably not been the most important selection pressure on
• 2.female human body shape.
• 3.2. Male humans in different cultures find different aspects of the female body attractive
• 4.and therefore are unlikely to have exerted consistent directional sexual selection on
• 5.the female body.
• 6.3. Breast size is not correlated with lactation success.
• 7.4. Visible hip width is not correlated with parturition success.
• 8.5. Women would lower their fitness if they tried to deceive men about their internal
• 9.pelvic dimensions.
• 10.6. There are many alternative hypothesis to explain the existence of fat onwomen's
• 11.breast, hips, and buttocks.

     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

On the purification and characterization of exopeptidases from antarctic krill,Euphausia superba

• 1.1. Two carboxypeptidase-A type of enzymes and two carboxypeptidase-B type of enzymes effecting hydrolysis of Hipp-l-Phe and Hipp-l-Arg respectively, have been purified from E. superba using gel filtration, affinity chromatography and FPLC-anion exchange chromatography. In addition an aminopeptidase has been partly purified.
• 2.2. The carboxypeptidases had mol. wts of 27,000 (carboxypeptidase A) and 31,000 (carboxypeptidase B).
• 3.3. Carboxypeptidase A exhibited a broad pH optimum with a maximum at pH 5.5–6.5, whereas carboxypeptidase B had a more narrow pH-optimum with a maximum at pH 7. The aminopeptidase had an optimum at about pH 8.7.
• 4.4. The carboxypeptidases were inhibited by the chelating agent 1,10-phenanthroline.

     

An enzyme-inhibitor complex of cathepsin L in the white muscle of chum salmon (Oncorhynchus keta) in spawning migration

• 1.1. A cathepsin L-inhibitor complex was purified from the white muscle of chum salmon (Oncorhynchus keta) by a series of chromatographies on DEAE-Sephadex, con A-Sepharose and Sephadex G-150.
• 2.2. The mol. wt of the complex was estimated to be 50,000 by gel filtration. The complex per se showed little activity of cathepsin L, but it became active when incubated at an acidic pH.
• 3.3. SDS-PAGE analysis and an experiment of activation by acidification indicated that the complex consisted of the 37 or 30 kDA-form of cathepsin L and the 15 kDa-endogenous cysteine protease inhibitor.
• 4.4. The enzyme-inhibitor complex was considered to be formed when cathepsin L leaks out of the lysosome in vivo or is freed from the lysosome when the tissue is artificially destroyed.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: Hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of tris

• 1.1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH.
• 2.2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint.
• 3.3. Individual constants of rate of a simple transphosphorylation mechanism have been determined.
• 4.4. At high Tris concentrations (> 1.0 M) a slight competitive inhibition has been observed.
• 5.5. Inhibition in NH₄⁺-NH₃Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH₂ group of Tris is responsible for inhibition.
• 6.6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.