A new spectrophotometric assay for protein in cell extracts

Kalb, Vernon F.; Bernlohr, Robert W.

doi:10.1016/0003-2697(77)90173-7

Cited by 624 publications

(197 citation statements)

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“…Peak areas were converted using the standard curve to amounts expressed as mmoles per bottle. Protein was quantified by an ultraviolet absorption method (Kalb and Bernlohr, 1977) following hot alkaline lysis of the cells (Coleman et al, 2002a). Chloride analysis was done via a colorimetric assay (Coleman et al, 2002b).…”

Section: Analytical Techniquesmentioning

confidence: 99%

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

Coleman

et al. 2011

The ISME Journal

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The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH 4 ) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH 4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C 2 -C 4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis-this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min -1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C 2 -C 4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.

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Section: Analytical Techniquesmentioning

confidence: 99%

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

Coleman

et al. 2011

The ISME Journal

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“…Spectrophotometric methods (Kalb & Bernlohr, 1977;Whitaker & Granum, 1980) were used for protein determination.…”

Section: Enzyme Assays Enzyme Activities Were Determined In Mmentioning

confidence: 99%

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

Chistoserdova

Lidstrom

1996

Microbiology

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A region on the Methylobacterium extorquens AM1 chromosome previously shown to complement a chemically induced mutant (PCT48) unable to convert acetyl-CoA into glyoxylate was characterized in detail in order to identify the gene(s) involved in the unknown pathway for acetyl-CoA oxidation. Six complete and two partial ORFs were identified by sequencing. Sequence comparisons suggested these might code for, respectively, a dehydrogenase of unknown specificity, a polypeptide of at least 15 kDa with unknown function, a coenzyme-B,,-linked mutase, a catalase, an alcohol dehydrogenase (ADH) of unknown function, a polypeptide of 28 kDa, a ketol-acid reductoisomerase and a propionyl-CoA carboxylase (PCC), Insertion mutations were introduced into each ORF in order to determine their involvement in C, and C, metabolism. Mutations in three genes, encoding the mutase, ADH and PCC, resulted in a phenotype characteristic of mutants unable to oxidize acetyl-CoA, i.e. they were C, -and C,-negative and their growth on these compounds was restored by the addition of glycolate or glyoxylate. Mutants in the genes thought to encode catalase and PCC were found to be deficient in the corresponding enzyme activity, confirming the identity of these genes, while physiological substrates for the mutase and ADH remain unidentified. This work, in which three new genes necessary for conversion of acetyl-CoA into glyoxylate were identified, is an intermediary step on the way to the solution of the unknown pathway for acetyl-CoA oxidation in isocitrate-lyase-negative methylotrophs.

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“…The spectroscopic method of Kalb and Bernlohr [16] was used for the concentration determination of crude halophilic proteins in a Zeiss PMQII (FRG) or Cary 11 8 Varian (USA) spectrophotometer.…”

Section: Concentration Determinationsmentioning

confidence: 99%

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 1. Conformation and interaction with Water and Salt between 5 M and 1 M NaCl Concentration

Pundak

Eisenberg

1981

Eur J Biochem

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Large-scale growth of extreme halophilic bacteria from the Dead Sea and purification of malate dehydrogenase (and other proteins) in quantities of hundreds of milligrams makes possible a detailed study of the adaptation to high salt. Halophilic malate dehydrogenase is stable at 20 "C in NaCl solutions between 2.5 -5 M. Below 2.5 M NaCl time-dependent inactivation, paralleled by structural changes, sets in. Within the time scale of the sedimentation, diffusion and circular dichroism experiments discussed here, it was possible to analyze data corresponding to the active halophilic malate dehydrogenase between 1 M and 5 M NaCl. The striking observation was that rather minor conformational changes were observed over the whole range, yet the special properties of the halophilic enzyme seem to be related to its capacity of associating with unusually large amounts of water and of salts, quite distinct from non-halophilic counterparts. These special properties seem to be related to the intact structure of the protein. Some parallel properties of halophilic glutamate dehydrogenase are also discussed.Extreme halophilic bacteria grow best in 20 -30 % NaCl 11 -31. These obligate halophiles are red-pigmented organisms which occur naturally inwaters of high salinity such as theDead Sea, for instance. The extreme halophilic bacteria adapt themIselves to life at high salinity by having internal concentrations (of salt that equal or exceed even the high concentrations in which they grow. They lyse at low salt concentrations. For recent reviews of the unusual properties of the halobacteria, some of which bear closer resemblance to eukaryotes rather than to prokaryotes, we refer to Kushner [4] and to Bailey ,md Morton [5]. Striking compositional properties, which have also been extensively studied in our laboratory are that halophilic proteins possess a molar excess of 16-18 % negatively charged amino acids [6 -81, whereas in non-halophilic proteins the excess is only 7-9%. The content of hydrophobic amino acids is significantly low, yet the content of borderline hydrophobic amino acids (e.g. alanine and threonine) is higher than in non-halophilic counterparts.Halophilic proteins carry out identical functions to their nonhalophilic counterparts, yet in a medium in which one parameter (salt concentration) differs greatly. The halophilic character is an intrinsic property of the proteins and does not depend on additional factors [7,9,10]. The proteins are stabilized by multimolar salt concentrations and the stabilization effect of different salts follows the Hofmeister lyotropic series. At high salt concentrations (3-5 M NaCl or KC1) enzymatic activity of halophilic proteins is observed whereas non-halophilic enzymes tend to be inactivated and to precipitate [11,12]. At low salt concentrations (< 2 M NaCI) halophilic enzymes denature. Denaturation involves unfolding, dissociation and inactivation, processes which may

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A new spectrophotometric assay for protein in cell extracts

Cited by 624 publications

References 19 publications

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 1. Conformation and interaction with Water and Salt between 5 M and 1 M NaCl Concentration

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