Solution behaviour of Chromobacter viscosum and Pseudomonas sp. lipases. No evidence of self-association.

1. The size of two bacterial lipases was studied by SDS/PAGE, sedimentation velocity and sedimentation equilibrium to test for possible self-association behaviour. 2. Mr values of selected lipases were obtained from SDS/PAGE and sedimentation-velocity measurements, together with an absolute determination by sedimentation equilibrium 3. The Mr values obtained in a variety of aqueous solvents indicate that lipases do not self-associate in solution, suggesting the absence of surface hydrophobic patches.

Preparation of fluorescent derivatives of lipases and their use in fluorescence energy transfer studies in hydrocarbon/water interfaces.

Fluorescein isothiocyanate reacted with a chromobacter and pseudomonad lipase to yield mono-substituted, fully active, enzymes. With the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in the non-aqueous phase, fluorescence energy transfer was used to follow the lipase and similarly labelled model proteins in and out of the interface in heptane, and heptane/di-O-palmitoyl-rac-glycerol (a substrate analogue), emulsions. Competitive binding, and displacement by other proteins could also be followed.

Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase.

Poly(A)+ RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A)+ RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approximately 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G X C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.

Stability of enzymes.

Enzymes can lose activity through covalent and noncovalent structure alterations. In the former, protease attack and modification by small active molecules such as oxygen are important. Conformational stability can be measured by Tm, the midpoint temperature of the thermal denaturation curve, and turnover in vivo of a number of enzymes correlates with Tm. Measurement of Tm and delta Cp leads to evaluation of delta H, T delta S, and delta G for the unfolding process. The importance of d(delta G)/dT is emphasized since it can be used to evaluate the temperature of maximum stability. There is no simple relationship between amino acid sequence and delta Gmax, nor can the effect of mutation be accurately forecast. Reversibility of folding is an important factor in stability. Tm correlates with [G]1/2, the midpoint guanidine unfolding concentration, and is the most useful predictive quantity for enzyme stability.

The interaction of mammalian medium-chain hydrolase with yeast fatty acid synthetase.

The interaction of rat mammary gland medium-chain thioesterase with yeast fatty acid synthetase has been investigated. Medium-chain thioesterase interacts with yeast fatty acid synthetase causing premature chain termination of the fatty acids synthesized from acetyl-CoA and malonyl-CoA. This effect is most marked under conditions of rate-limiting malonyl-CoA availability. Immobilized yeast fatty acid synthetase specifically binds rat mammary gland medium-chain thioesterase. This interaction has been used to purify medium-chain thioesterase to near homogeneity from samples of rat mammary gland cytosol. The stoichiometry of binding of medium-chain thioesterase to yeast fatty acid synthetase has been investigated. Yeast fatty acid synthetase binds 5.7 +/- 1 mol medium-chain thioesterase/mol yeast fatty acid synthetase. It is concluded that yeast fatty acid synthetase has a medium-chain thioesterase binding site.

Stability and inhibition of Aspergillus and Rhizopus lipases.

A lipase (triacylglyceral aclhydrolase, EC 3.1.1.3) secreted by Aspergillus contains two chains each of molecular weight 25000, with 12 mannose, two galactose and two N-acetylglucosamine residues per chain. The average hydrophobicity is close to the mean fov globular proteins. Thermal denaturation was biphasic when followed by circular dichroism but the stability is not unusual for globular proteins, nor does partial removal of the carbohydrate affect it. (delta G=46kJ). Refolding did not occur. The inhibitory effect of 2H2O on the rate of reaction implicates a histidine in the active centre. The inhibitory effect of concanavalin A was complex but the active centre was blocked. A lipase from Rhizopus arrhizus showed immunological identity with Aspergillus lipase, but unlike that one, was not inhibited by the antiserum. Similarly it was not inhibited by concanavalin -A although Con A-Sepharose was used in its isolation. It was 4-fold as active as the Aspergillus enzyme but had low thermal stability (delta K= 16kJ). The interfacial location of lipases does not impose any special requirement on the overall structure of the enzyme.

Myofibrils, sarcoplasmic reticulum and phosphorylase kinase of Pietrain and Large White-landrace pigs.

Comparison of fast and slow twitch myofibril preparations by SDS-gel electrophoresis showed no significant differences between Large White and Pietrain pigs. No variants of parvalbumins or calsequestrin (calcium binding proteins) were found. Pietrain phosphorylase kinase has a greater stimulating activity on porcine phosphorylase than does Large White phosphorylase kinase.

A determination of the sub-units of arachin by osmometry. Arachins A, B, and A1.

1. Osmotic pressure determinations of dissociated arachins are a particularly suitable method for determination of the number of sub-units in the protein, because they yield a number-average molecular weight. 2. Arachin, in 8m-urea-0.1m-sulphite, produces 12 sub-units from the form of molecular weight 345000. 3. When the urea concentration is varied the molecules became fully dissociated at 6m-urea-0.1m-sulphite. Although sulphite is necessary to break disulphide bridges, concentrations greater than 0.1m cause a re-aggregation of the sub-units. Similar results were obtained in guanidine solutions. 4. A new form of arachin has been discovered, A1, migrating more rapidly than arachin A. 5. The N-terminal residues of arachin have been re-investigated on more highly purified samples: they are glycine, valine and (iso)leucine in the proportions 4:1:1. 6. The three forms of arachin have the structure (B) beta(4)gammadelta, (A) alpha(2)beta(2)gammadelta and (A1) alpha(4)gammadelta, for the forms of molecular weight 170000. 7. Dissociation in 8m-urea produces some fragments, detected by gel electrophoresis, which appear to be dimers of the type alpha-S-S-beta, beta-S-S-beta, held together by disulphide bonds.

Protein bodies of the soybean.

Some microscope observations of the protein bodies of the cotyledon cells of the soybean (Glycine max) are described, together with changes in their appearance which occur on germination. Density gradient centrifugation permits the isolation of protein bodies from soymeal. They contain about 70% of the protein of the bean. Only 1 protein could be detected in them: glycinin, the major soybean protein.The protein bodies were fractionated to light and heavy fractions. The former contained 97.5% protein, the latter 78.5%. RNA, phytic acid and lipids were also present. The 2 fractions probably differ only in the extent of contamination by other cell fragments.