To establish a covalent chromatography system for purification of naturally occurring chymotrypsin-like serine proteases, a diphenyl 1-amino-2-phenylethylphosphonate derivative bearing Gly-Gly-Gly as a spacer was immobilized on the Sepharose 4FF gel. In this system, bovine chymotrypsin was selectively bound to the phosphonate immobilized on the gel and then released by the action of 2-pyridinealdoxime methiodide as the reactivated enzyme. Based on the study results for selective binding and reactivation conditions, chymotrypsin-like proteases from pancreatin (hog pancreas) were rapidly and highly purified within three hours.

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Escherichia coli multiple-deletion mutants were constructed to examine the lipid phosphatase genes. Ratio of cellular levels of undecaprenyl phosphate (UP) and undecaprenyl diphosphate (UPP) was determined using cells labeled with 14C and measuring the radioactivities in these lipids. The labeled cells were obtained through feeding [14C]isopentenyl diphosphate to an E. coli strain. The relative level of UP in the wild type strain W3110; in the YS1234 strain defective in ynbD, yeiU, ybjG, and pgpB; in the YS1235 strain defective in ynbD, yeiU, ybjG, and bacA; and in the YS1245 strain defective in ynbD, yeiU, pgpB, and bacA were 78.0% ± 3.5%, 74.7% ± 1.2%, 68.4% ± 6.8%, and 64.0% ± 7.5%, respectively. The quadruple mutant strain that possessed the wild-type bacA showed almost identical UP level as the wild type strain W3110, indicating that sufficient level of UPP phosphatse activity was conferred by a bacA product without the products of ynbD, yeiU, ybjG, and pgpB. However, the mutants defective in bacA showed a slight but significant (p 0.01) decrease in the UP level.

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The proteins in human hair consist primarily of microfibrillar keratins with a molecular mass of 40–65 kDa and keratin-associated proteins (KAPs) with a molecular mass of 6–30 kDa, according to the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Because an effective purification procedure of KAPs has not been established, little is known about the protein chemistry of KAPs as compared with that of keratin. When hair samples were incubated in the Shindai solution containing alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-methyl-1-propanol, the extraction of KAPs was enhanced, while extraction of keratin was suppressed. Using ethanol, we established a selective purification procedure for KAPs and keratin. According to Tricine/SDS-PAGE, the KAPs fraction contained six polypeptides with molecular masses of 3.5, 4.4, 5.2, 7.8, 15, and 28 kDa. The keratin fraction contained two polypeptides with molecular masses of 45 and 67 kDa and was free of low-molecular-weight components. The amino acid compositions of the KAPs and keratin fractions were mostly in agreement with the values found in the literature. The recoveries of the KAPs and keratin fractions from the hair samples were approximately 10 and 50%, respectively. Scanning electron microscopy (SEM) showed that hair samples retained fine fibrous structures in the cortex after extracting the KAPs and that the additional extraction of keratin caused the fibrous structures to disappear. These results indicated that KAPs may function by surrounding the fibrous structures and supporting the keratin fibers in the cortex. In this study, we propose a novel and convenient isolation procedure for KAPs and keratin from human hair.

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