5'-Deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase was purified to homogeneity from Propionibacterium shermanii by a simplified procedure. The native enzyme has an apparent Mr of 165,000, similar to the enzyme from other sources but larger than previously reported. It consists of two non-identical subunits, of Mr 79,000 and 67,000 respectively. The smaller subunit is apparently not a proteolytic fragment of the larger one. The final preparation usually contained some inactive mutase, bearing a tenaciously bound cobalamin species. This protein proved to be readily separable from apoenzyme by fast protein liquid chromatography on anion-exchange columns.

The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.

RbstractW e have developed a highly sensitive and specific immunoassay for humanlrat corticotrophin-releasing factor-41 (CRF-41) to enable determination of immunoreactive CRF-41 levels in biological samples. To achieve high specificity, sensitivity and s p e e dwe have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay h a s a sensitivity of 0.08 fmollwell compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF-41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF-41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF-41 coeluting with humanlrat CRF-41 and its oxidized form Met [02'~38]CRF-41 in human and rat tissue extracts separated by high-performance liquid chromatography. The ability to measure immunoreactive CRF-41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.( 'orticotrophin-releasing factor-41 (CRF-41) is a 41 amino-acid pcptide originally isolated from ovine hypothalamic tissue ( I ) . 7hc amino-acid sequences of human and rat CRF-41 are identical and differ from the ovine sequence at 7 residues (2, 3). CRF-41 cilso shares considerable sequence homology with sauvagine, a fi.ogskin peptide which has corticotrophin-releasing activity (4). 1,'arly assays for C R F relied on bioassay techniques ( 5 ) and were, therefore, susceptible to interfercnce from substances such as :idrenaline or arginine vasopressin which also have corticotrophinlcleasing activity (4). The development of RIAs for CRF-41 (6) allowed more specific and sensitive assay. However, the RIA tcchnique is time consuming and can give elevated measurements in the presence of CRF-41 peptide fragments or peptides with dcgrees of sequence homology with CRF-41 (4). Such RIAs also 1-cquire extraction of the material from biological fluids to reduce interference (7-9). The development of a 'two-site' immunoradiometric assay (IRMA) for CRF-41 (10) was a significant advance, reducing interference from fragments and allowing direct measurement of the peptide in biological fluids including plasma (1 1) In this study, we sought to construct an enzyme amplified ilnmunometric assay (EAIA) for human/rat CRF-41 using two polyclonal antisera raised against the whole CRF-41 molecule. The technique uses one antibody bound to a solid phase with sequential addition of samples, second antibody-enzyme conjugate and substrate. The assay was based on the technique of enzyme amplification (12) to improve sensitivity and reduce assay time (13). The performance of this assay has been compared t o two RIAs for CRF-41 in the measurement of rat hypothalamic CRF-41 content and release. The ability of the EAIA to measure human CRF-41 in both unextracted gest...

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