Gordon T. James

Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers

Abstract Aqueous preparations of phenylmethylsulfonyl fluoride (PMSF) become inactive toward proteases unless promptly brought into contact with protease. Inactivation of PMSF increases with increased pH and temperature. Half-lives of the inhibitor at 25°C are approximately 110,55, and 35 min at pH 7.0, 7.5, and 8.0, respectively. At pH 8, 100 μ m PMSF is almost completely inactivated within 1 hr at 25°C or within 22 hr at 4°C. Stock solutions of PMSF in 100% isopropanol are stable at 25°C for months if not longer. Reactivation of PMSF-inhibited chymotrypsin did not occur within 1 week at 25°C at pH 7.0.

Studies in metachromatic leukodystrophy. XIV. Purification and subunit structure of human liver arylsulfatase a

Arylsulfatase A was purified to apparent homogeneity from normal human livers obtained at autopsy. According to gel electrophoresis in sodium dodecyl sulfate, purified arylsulfatase A consistently contained two subunits of slightly different sizes: approximately 69 000 and 57 000 daltons, but were not present in stoichiometrically equal amounts. Peptide maps of the entire enzyme and of the two individual subunits showed that the two polypeptides share similar if not identical sequences. These observations raise the possibility that the smaller polypeptide might be derived from the larger one. The sensitive peptide mapping procedures employed will make feasible future studies with the abnormal enzyme found in metachromatic leukodystrophy.

Essential arginine residues in human liver arylsulfatase A.

Abstract Human liver arylsulfatase A was treated with arginine-specific reagents (diones), resulting in a loss of enzyme activitity with apparent first-order kinetics. Sulfite and borate—competitive inhibitors of the enzyme—provided complete protection from inactivation by phenylglyoxal. Sulfite and substrate each likewise protected against enzyme inactivation by 2,3-butanedione. A plot of pseudo-first-order rate constants of enzyme inactivation versus 2,3-butanedione concentrations suggests that an essential arginine residue is modified with a loss in function of the binding site or of the active site of the protein. Chemical analysis of the butanedione-treated sulfatase indicates that complete enzyme inactivation corresponds to a modification of only about 2 of the 20 arginine residues per enzyme subunit. Taken together, all of the results strongly suggest that arginine residues are essential for the activity of arylsulfatase A. An incidental discovery in this work is that borate ion is a competitive inhibitor of human arylsulfatase A with a Ki of 2.5 × 10−4 M.

Separation of leukocyte peroxidase isoenzymes by agarose-acrylamide disc electrophoresis

Two methods for releasing peroxidases from cellular membranes were compared. One method using EDTA/HTAB to release total enzyme yielded 10–20 times more activity than another method using Cetrimide and Brij-35. Electrophoresis on agarose-acrylamide gels showed four cathodic isoenzymes (A, B, C, and D) from the EDTA/HTAB preparation, but only isoenzyme B was seen from the Cetrimide-Brij preparation. This B isoenzyme was the major band of activity in both preparations, except in elderly patients.Peroxidase isoenzymes were stable to heating at 50°C for up to one hour. However, at 75°C, only the major B isoenzyme remained stable. Aminotriazole and PHMB did not reduce the activity of isoenzyme B; however, the others were completely inhibited. Sodium azide at 10 mM inhibited all isoenzymes.Human leukocyte isoenzyme patterns appear to shift with age. Thus, young controls (23–38 yr.) had most of their total activity (70+%) residing in the isoenzyme B band. The fastest moving cathodic isoenzyme, D, accounted for only 15%. However, with advancing age, the amount of isoenzyme B activity declined whereas that of isoenzyme D increased.Thus, in 6 out of 7 normal older controls; age 60–80 years, the relative amounts of isoenzyme B and D were approximately equal. Moreover, in one 97-year-old woman, the D isoenzyme was twice as much as the B isoenzyme.Technical differences in the methods of preparation and in the resulting isoenzyme patterns can explain certain of the apparent discrepancies in the literature regarding decreased levels of leukocyte peroxidase in neuronal ceroid-lipofuscinosis. For example, when peroxidase activity was studied in neuronal ceroid-lipofuscinosis, using the Cetrimide-Brij method, isoenzyme B was being measured, not other isoenzymes.

Nuclear Magnetic Resonance (Nmr)

The extent of this chapter parallels the widespread acceptance of nmr as a tool for biochemical investigation (see the series Biological Magnetic Resonance by Berliner and Reuben, 1978; Wasson, 1984; James, 1975; Dwek, 1973). For example, if one examines current issues of a journal such as Biochemistry it is scarcely possible to find one which does not contain at least one biological nmr paper. The spectroscopic method described here is known as high-resolution nmr, and as such is a powerful tool for the elucidation of chemical structures in solution. More recently it was discovered that high-resolution spectra may also be obtained from solids and liquid crystals, something that was not anticipated in the early period. Thus, the method is particularly applicable to cellular components, which range from dissolved entities such as proteins and intermediary metabolites to more condensed phases of the type made up of phospholipids and sterols in biological membranes.

Circular Dichroism (CD) and Magnetic Circular Dichroism (MCD)

For reasons given in Chapter 7, ultraviolet and visible light absorption measurements offer very little information of a selective nature. Two other methods which use light in this wavelength range, optical rotatory dispersion (ORD) and circular dichroism (CD), provide evidence of a structural/stereochemical nature, although to gain such insight it is necessary to compare the results from ORD and CD with information from other methods. When the sample under investigation is subjected to an intense magnetic field (-50 kG) the phenomena observed in ORD and CD are extended, respectively, to magnetic optical rotatory dispersion (MORD) and magnetic circular dichroism (MCD). This chapter is concerned mostly with MCD. However, before attempting a discussion of MCD it will be necessary to first present a brief account of the basic characteristics of the related methods (Djerassi et al., 1971; Stephens, 1974).

Bioinorganic Topochemistry: Microprobe Methods of Analysis

Direct elemental ultramicroanalysis of biological samples is now possible, based on X-ray emission stimulated by a highly focused electron beam. This is an elegant extension of the X-ray spectrometric identification and quantitation method for specific elements. The method is made possible by combining either an energy- or a wavelength-dispersive X-ray spectrometer with a scanning electron microscope. The hybrid is appropriately described as a tool for inorganic topochemistry, since the beam may impinge upon a resolved structural feature while the X-ray emission spectrum is recorded. This procedure is especially valuable in that all of the elements heavier than beryllium may be specifically determined. Figure 11-1 presents a schematic representation on a typical electron microprobe instrument.

Laser Applications: Resonance Raman (RR) Spectroscopy and Related Methods

An application of laser technology to the detection of quadrupole resonance phenomena was described in Chapter 3 (see Section 3.4.2). The present chapter explores resonance Raman (RR) spectroscopy. It should be noted that RR spectroscopy is but one of many potential applications involving lasers (Brewer and Mooradian, 1974; Bradersen, 1979; Morris and Wallan, 1979; Butler et al., 1978; Omenotto, 1979; Steinfeld, 1978; Horrocks et al., 1980; Parker, 1983); yet it does have the distinction of providing information of a relatively selective nature. Also, it is the one biological application of lasers which has been used sufficiently to justify its inclusion as a whole chapter. Raman and RR spectroscopy are relatively old methods with biological applications extending back to the 1930s. The laser improvements are most recent.

Neutron activation analysis.

The discovery of the neutron by Chadwick in 1932 is a relatively recent event in the history of science (see Heller, 1976). Soon after that discovery, neturons had been used to activate elements, and Bowen (1956) showed that neutron activation could be a highly sensitive method of analysis.

Nuclear Quadrupole Resonance (Nqr)

This brief chapter has been included in the anticipation that biological applications of nqr spectroscopy will increase as time passes. As in the other spectroscopic methods based on nuclear properties, nqr is able to probe the electronic environment at a specific type of nucleus (Smith, 1978; Semin et al., 1975; Schultz, 1970). It is a solid-state phenomenon and thus has much in common with the Mossbauer effect, while biological nmr is oriented largely toward the liquid and liquid-crystalline states. To date, most nqr investigations have dealt with fairly simple substances. The method is generally applicable to the study of a variety of elements (Biryukov and Vorokonov, 1972), and in spite of certain limitations, biological applications may be expected to increase. For the present, however, small molecules are more appropriate subjects for nqr.