Bengt v. Hofsten

Oxygen uptake of bottom sediments studied in situ and in the laboratory

Abstract Oxygen uptake by soft bottom sediments was measured in situ with an oxygen electrode in a bell jar. Values in the range 0·3-3·0 g O 2 m −2 d −1 were obtained at 19 localities in fresh and brackish water. Comparative measurements were made in the laboratory on sediment cores. These gave consistently lower values than the in situ measurements. Laboratory experiments showed that the oxygen uptake depended on the oxygen concentration and that the temperature coefficient decreased with increasing temperature. There was no simple correlation between oxygen uptake and content organic matter in sediments.

An extracellular proteolytic enzyme from a strain of Arthrobacter II. Purification and chemical properties of the enzyme

Summary 1. A new extracellular proteolytic enzyme has been isolated from the culture medium of a strain of Arthrobacter grown in the presence of gelatin. The fractionation procedure makes use of precipitation with ammonium sulfate in the presence of cellulose, treatment with DEAE-Sephadex, gel filtration on Sephadex G-100 and crystallisation with ammonium sulfate or acetone. 2. Ultracentrifugation of the crystallized enzyme showed only one sedimenting component and its molecular weight determined by equilibrium centrifugation was approx. 22 ooo. Free electrophoresis revealed only one ultraviolet-absorbing peak with a mobility of 2.57 · 10−5 cm2 · V−1 · sec−1 at pH 8.6. 3. Twice-crystallized enzyme may contain traces of peptides which can be removed by gel filtration in aqueous acetic acid. Some of the pitfalls encountered in studies on the amino acid composition of the enzyme are discussed. 4. The enzyme contained 221 amino acid residues with a formula weight of 23 041. The amino-terminal residue was valine. The protein contains 2 disulfide bridges, a total of 12 basic amino acid residues and 29 amide groups. The molar extinction coefficient is 44.4 · 103 reflecting a high content of tyrosine and tryptophan.

Immunological precipitates in agarose gels

Abstract Comparative studies of immunological precipitation reactions in ordinary agar and agarose gels have been made. The strong electroendoosmosis obtained in agar during immunoelectrophoresis is strongly reduced in the uncharged agarose gel. Agarose was also found to give more distinct lines of precipitation in gel diffusion analysis of basic antigens, and no halo effects or false precipitation lines were observed with this product.

Purification and some properties of an acid phosphatase from Escherichia coli.

Abstract 1. 1. Escherichia coli contains several enzyme components which catalyze the hydrolysis of phosphate esters at an acid pH. One of these components has been purified by means of chromatography, zone electrophoresis and gel filtration to such an extent that it appeared to be essentially free from contaminating material. 2. 2. The enzyme, which is active against hexose monophosphate and phenyl monophosphate esters, is remarkably stable both in 1 M acetic acid in the absence of salts and in 0.2 M Na 2 CO 3 . 3. 3. Zone electrophoresis in Tris-HCl (pH 8.2) of a cell-free extract as well as of partially purified preparations of the enzyme gave three protein fractions with acid phosphatase activity. The most rapidly migrating of these represented the major part of enzyme activity and was further purified by gel filtration on Sephadex G-75 in 1 M acetic acid. 4. 4. The fractionation obtained on the Sephadex and sedimentation studies indicate that the monomeric form of the enzyme has a molecular weight of less than 20 000. Concentration of enzyme solutions, both in 1 M acetic acid and in less acidic solutions, leads to polymerization. 5. 5. Preliminary data are given on the amino acid composition of the purified enzyme. The minimal molecular weight has been estimated to be 13 000–14 000.

An extracellular proteolytic enzyme from a strain of Arthrobacter I. Formation of the enzyme and isolation of mutant strains without proteolytic activity

Summary 1. A strain of Arthrobacter isolated from a grass infusion has been shown to produce a proteinase during growth in media containing peptides and proteins. The kinetics of the enzyme formation show that the enzyme is truly extracellular and almost no enzyme activity is associated with the cells. 2. Certain sources or carbon for growth, such as carbohydrates and some amino acids, repress markedly the proteinase formation. It is concluded that a non-repressing growth environment is a necessary but probably not sufficient condition for a high rate of proteinase synthesis. Proteins and peptides probably play a role in the induction or excretion process. 3. Mutant strains which are unable to excrete extracellular proteinase have been isolated by use of an agar medium containing milk to indicate the proteolytic activity of bacterial colonies.

Acid phosphatase and the growth of Escherichia coli.

Abstract 1. 1. The acid phosphatase activity of E. coli varies considerably with the conditions of growth and the physiological state of the cells. The nature and concentration of the source of phosphate for growth has no marked influence on the formation of acid phosphatase in contrast to the alkaline phosphatase. It was found that succinate or glycerol as the source of carbon for growth supported the synthesis of high levels of acid phosphatase, whereas carbohydrates have a repressive effect. 2. 2. Certain properties of the enzyme activity as measured by the rate of hydrolysis of p -nitrophenylphosphate at pH 4.7 have been studied. The enzyme does not seem to be firmly bound to a particulate fraction of the cells. Some of the possible functions of the activity are briefly discussed.

Purification and metal ion activation of an aminopeptidase (aminopeptidase II) from Bacillus stearothermophilus

Abstract 1. 1. One of the aminopeptidases (aminopeptidase II) of the obligate thermophilic bacterium Bacillus stearothermophilus has been purified by gel filtration and chromatography on DEAE-Sephadex and SE-Sephadex. Its molecular weight and isoelectric point are 80 000–100 000 and 4.3, respectively. 2. 2. The enzyme is located in the cytoplasm of the bacterium, which also contains a carboxypeptidase and a d -aminopeptidase. 3. 3. Aminopeptidase II is a metal ion (M2+)-dependent enzyme, and no ions other than Co2+ or Zn2+ were found to give an active ESM2+ complex with the substrate l - leucine -p- nitroanilide . 4. 4. Kinetic data show that with Co2+ the formation of the ESM2+ complex is a 2-step process with the substrate molecule binding only to the ECo2+ complex. This activation is very fast. 5. 5. In the presence of both Mn2+ and Co2+, an inactive ESMn2+ complex can be formed. Kinetic experiments indicate separate binding sites for the metal ion and the substrate molecule. 6. 6. A lag in the reaction between apoenzyme and substrate was observed at low concentrations of Co2+ and Zn2+. Beside being involved in the hydrolysis of the substrate, these ions probably bring about a structural change in the enzyme protein. Other ions such as Mn2+, Cd2+ and Ni2+ can also activate the apoenzyme in a similar way without having a catalytic function.

Estimating the rate of degradation of cellulose fibers in water

A method is described by which the rate of degradation of different kinds of cellulose fibers was determined in aquatic environments. The fiber samples were enclosed in small bags of nylon fabric with a fine mesh and weight losses of bags placed in water or sediments were measured over periods of several months. Newly processed pulp fibers were rapidly degraded in aerobic, nutrient-rich water whereas previously dried pulp and fibers derived from cotton were degraded more slowly. Lignin-containing mechanical pulp was hardly degraded at all. Cellulose decomposition was slow in unpolluted sea water and in the heavily polluted, anaerobic water outside a paper mill but relatively rapid both in the anaerobic water and the sediment of a nutrient-rich lake. The applications and limitations of the bag method in studies of decomposition processes in aquatic ecosystems are discussed.

An extracellular proteolytic enzyme from a strain of Arthrobacter III. Stability and substrate specificity

Summary 1. The stability and enzymatic properties of a crystallized proteinase isolated from the culture medium of an Arthrobacter strain have been studied. The enzyme is stable for hours at 37° in 10−2 M HCl, 1 M acetic acid and in buffers up to pH 11. The rate of autodigestion is low under most conditions and the enzyme is active over a wide pH range. There is no indication of a dependence on metal ions for stability and activity; no free SH-groups could be detected in the enzyme. 2. The hydrolysis of native and denatured proteins has been studied by following the release of trichloroacetic acid-soluble material and ninhydrin positive compounds and by pH-stat experiments. The specificity of the enzyme is broad, but free amino acids are usually not liberated. At least ten different peptides were formed from the B chain of oxidized insulin and about 50% of the bonds in oxidized ribonuclease were hydrolyzed. 3. Poly- L -lysine was hydrolyzed to peptides containing two or more lysine residues. Certain synthetic peptides, which were not hydrolyzed by the proteinase were hydrolyzed by extracts from the cells. N-Benzoyl- L -arginine ethyl ester and other esterase substrates were hydrolyzed. Incubation of the enzyme with 10−3 M diisopropyl phosphofiuoridate completely abolished the enzyme activity.

The inhibitory effect of galactosides on the growth of Escherichia coli.

Abstract 1. 1. The growth of strains of Escherichia coli that possess β-galactoside permease and synthesize β-galactosidase at a high rate is temporarily inhibited by the addition of lactose. Cultures growing on succinate as the source of carbon are particularly sensitive, and the inhibition is reversed by compounds that block the permease or enzyme activity. 2. 2. A number of other galactosides have been tested for their effect on the growth of various mutant strains of E. coli . 3. 3. The data presented indicate that the growth inhibitory effect is due to the accumulation within the cells of high concentrations of galactose, which may effect both the metabolism and the osmotic properties of the cells. 4. 4. Evidence is presented to show that galactose and structurally related compounds may be inhibitory as such withouth being phosphorylated to galactose 1-phosphate.