The reconstitution of lactic dehydrogenase after dissociation in 6 M guanidine hydrochloride or at acidic pH leads back to active tetramers indistinguishable from the native enzyme. In addition, aggregates of "irreversibly denatured" enzyme are formed to a certain extent. It has been shown that the ratio of both fractions depends on such factors as the extent of denaturation and the concentration of the enzyme in the process of reconstitution. The electron microscopical analysis of the aggregates shows a broad distribution of high molecular weight particles ( M , >lo6). As suggested by circular dichroism measurements in the far-UV and by degradation in strong denaturants, these particles are composed of individual monomeric chains with partially restored secondary structure. The aggregates are stabilized by noncovalent interactions. For determination of the factors responsible for the competition %e great majority of regulatory enzymes represents oligomers composed of subunits which are attached to each other by noncovalent interactions. As a consequence, reconstitution of oligomeric enzymes after dissociation with denaturants, such as guanidine hydrochloride or acid, involves folding and association. In the case of monomeric enzymes, the folding transitions occurring during the formation of the three-dimensional structure have been analyzed in great detail by denaturation-renaturation studies in vitro (Anfinsen & Scheraga, 1975). On the other hand, the reactions involved in oligomer formation are far less characterized (Jaenicke, 1979).The acquisition of the quaternary structure of a given enzyme comprises highly specific association reactions which must be subtly interconnected with folding transitions. Like the unfolding of monomeric enzymes, the dissociation and unfolding of oligomers have been shown to be reversible in many cases. Therefore, the parameters determining the in vitro refolding and reassociation can be analyzed in a quantitative way by transferring denatured and dissociated enzymes to quasi-physiological conditions. Under optimum conditions of denaturation-renaturation, yields close to 100% can be achieved, corresponding to complete reassociation, refolding, and reactivation to the native state of the enzyme. It has been previously shown for a number of enzymes that the product of reconstitution is indistinguishable from the initial native enzyme as far as the hydrodynamic, spectroscopic, and enzymatic properties are concerned (Jaenicke et al., 1975. In cases where only partial reactivation is observed, the latter statement holds only if "irreversibly denatured" material is separated from the native enzyme . No of reactivation and aggregate formation, the kinetics of aggregation were analyzed by stopped-flow laser light scattering experiments and compared with the kinetics of reactivation [cf. Rudolph, R., & Jaenicke, R. (1976) Eur. J. Biochem. 63, 409-4171. Aggregation is determined by a process with a reaction order greater than 2, competing with a fast first-order folding reaction in the path...

SummaryBlood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin γ-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin® P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor Xm complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XMa, were used as hybridization probes. Of 0.36 x 106 independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XMa-specific. sequences. The total length of the isolated factor XILIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.

The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription.

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