The ACB1 gene encoding the acyl-CoA-binding protein (ACBP) was disrupted in Saccharomyces cerevisiae. The disruption did not affect the growth rate on glucose but reduced the growth rate on ethanol slightly. Although the growth rate of the acb1-disrupted cells was unaffected or only slightly affected, the acb1-disrupted strain was unable to compete with wild type cells when grown in mixed culture. The acyl-CoA level in the disrupted cells was increased from 1.5- to 2.5-fold during exponential growth. The increase in the acyl-CoA level was caused solely by an increase in de novo synthesized stearoyl-CoA. Experiments with purified yeast fatty acid synthetase show that it will synthesize long chain acyl-CoAs in the absence of acyl-CoA-binding protein. The addition of ACBP to the incubation medium resulted in a dramatic decrease in the chain length of the synthesized acyl-CoA esters. Despite the fact that the stearoyl-CoA concentration was increased 7-fold and the Delta9-desaturase mRNA level was increased 3-fold, the synthesis of oleic acid was unchanged in the acb1-disrupted strain. The results strongly indicate that ACBP in yeast is involved in the transport of newly synthesized acyl-CoA esters from the fatty acid synthetase to acyl-CoA-consuming processes.

Cellulose is the major polysaccharide component of plant cell walls and is the most abundant organic compound on the planet. A number of bacterial and fungal organisms can use cellulose as a food source, possessing cellulases (cellobiohydrolases and endoglucanases) that can catalyse the hydrolysis of the beta-(1,4) glycosidic bonds. They can be classified into seven distinct families. The three-dimensional structures of members of two of these families are known. Here we report the structure of a third cellulase, endoglucanase V, whose sequence is not represented in any of the above families. The enzyme is structurally distinct from the previously determined cellulases but is similar to a recently characterized plant defence protein. The active site region resembles that of lysozyme, despite the lack of structural similarity between these two enzymes.

Using the tyrosine fluorescence quenching as a criterion for acyl-CoA binding, we have shown that acyl-CoA-binding protein (ACBP) binds acyl-CoA esters with a chain length greater than C8 with equal affinity. The binding studies indicated a binding stoichiometry of 1 mol of acyl-CoA/2 mol of ACBP. The protein was found in liver, adipose tissue, intestinal mucosa, kidney, heart, brain, muscles and mammary gland. The highest concentration was found in liver cytosol and the lowest in muscles and mammary gland. ACBP could not be shown to bind non-esterified fatty acids.

Acyl-CoA-binding protein from bovine liver was purified with the use of reverse-phase h.p.l.c. in the final step. The complete amino acid sequence was determined by using a combination of gas-phase Edman degradation and electron-impact and fast-atom-bombardment mass spectrometry. The sequence was confirmed by determination of the Mr by plasma-desorption time-of-flight mass spectrometry.

By the use of recombinant technology, several stable Chinese hamster ovary (CHO) cell lines expressing human FVIII were established. Thrombin treatment and SDS-PAGE analysis of the purified recombinant FVIII (rFVIII) revealed a striking difference from plasma-derived FVIII (pFVIII). A 43-kDa fragment of the FVIII heavy chain appears as a double band from rFVIII, while a single band from pFVIII is observed. All other fragments from the two samples appeared similar by SDS-PAGE. The heterogeneity is caused by incomplete tyrosine sulfation of one or more of the three potential tyrosine sulfation sites (Tyr718, Tyr719, Tyr723). To investigate if there is a general limitation and heterogeneity in the tyrosine sulfation of rFVIII, two other potential tyrosine sulfation sites on the FVIII light chain (Tyr1664, Tyr1680) were analyzed. The results show that both sites on the pFVIII light chain and on the rFVIII light chain are completely sulfated. The limitation of CHO cells to tyrosine sulfate rFVIII is therefore only restricted to a few sites. The two sulfated forms of rFVIII can easily be separated by ion-exchange chromatography, indicating the importance of the sulfate groups on the charge and/or conformation of FVIII. Both forms of rFVIII possess identical in vitro coagulation activity, von Willebrand factor binding, and thrombin activation profile. However, the difference in tyrosine sulfation may change other biological properties of FVIII.