Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain.

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate).

Conformational activation of acylpapains and acylcathepsin B's: resonance Raman and kinetic evidence.

It is shown that, by extending the size of the substrate to meet papain's specificity requirement, conformational changes occur in bonds adjacent to the scissile linkage in the acyl enzyme. The resonance Raman (RR) spectra of the dithioacylpapains CH3OC(=O)-Phe-NHCH2C(=S)S-papain, CH3O(=O))-Gly-Phe-NHCH2C(=S)S-papain, and CH3OC(=O)-Gly-Gly-Phe-NHCH2C(=S)S-papain, formed from thiono ester substrates, are found to be very similar, indicating that each dithioacyl enzyme takes up an identical B-type conformation in the -NHCH2C(=S)SCH2-linkages. Moreover, the kcat and kcat/Km parameters for the reactions involving these intermediates are similar (in the ranges 0.

Focal myositis and elevated creatine kinase levels in a patient with phaeochromocytoma.

A case of phaeochromocytoma with marked transient elevation of creatine kinase levels is presented. No obvious cause for the elevation was found in life, but on autopsy a non-specific focal myositis was discovered. Possible reasons for the raised creatine kinase levels and focal myositis are discussed.

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.

Determination of salbutamol in human plasma and urine by high-performance thin-layer chromatography.

A rapid, accurate and sensitive method for the determination of salbutamol in plasma and urine is described. Salbutamol is extracted using solid-phase techniques and converted to an indoaniline dye by reaction with dimethyl-p-phenylenediamine. The indoaniline is separated using high-performance thin-layer chromatography and quantified by absorption microdensitometry at 650 nm.

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters.

The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency.

Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy.

Resonance Raman spectroscopic data provide conclusive evidence for the existence of an acyl-enzyme intermediate during the reaction of a thionoester substrate, N-methyloxycarbonylphenylalanylglycine methyl thionoester (CH3OC(=O)-Phe-NHCH2C(=S) OCH3), with cathepsin B from porcine spleen. The resonance Raman spectrum of CH3OC(=O)-Phe-NHCH2C(=S)S-cathepsin B, where the thiol S is from the active-site cysteine residue, is compared to that of the corresponding papain acyl-enzyme. Within the limits of experimental error (+/-2 cm-1 for peak positions), there are no detectable spectral differences.

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.

Doppler power frequency spectrum analysis in the diagnosis of carotid artery disease.

Power Frequency Spectral Analysis (PFSA) which evaluates the power of the frequency components at a selected interval, was applied in this preliminary study using a 5MHz Doppler continuous wave flow velocity meter and a modified Edwards Spectraview 500 analyzer. PFSA was used to study the common, internal and external carotid arteries of ten young adult control subjects and 31 patients with angiographically documented carotid bifurcation lesions. Two useful indices are described: (1) the frequency band width at 50% of maximum power (f50%) and (2) the highest frequency recorded (fmax).

Assessing the reliability of measurements from the Krusen limb load monitor to analyze temporal and loading characteristics of normal gait.

This study assessed the reliability of measurements made by four physical therapists on healthy subject gait data recorded from the Krusen limb load monitor. The five components of step (stance time, time up, time to second peak, and force at the first and second peaks) were analyzed. Six components contributing to gait (ambulation time; velocity; cadence; average swing phase duration, left lower extremity; average swing phase duration, right lower extremity; and ratio of unilateral weight bearing, right lower extremity to left lower extremity) were also analyzed.

Epidemiology of penicillinase-producing Neisseria gonorrhoeae in Liverpool from 1977 to 1982.

After the 1976 outbreak of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections had been controlled, less than 1 per cent of cases of gonorrhoea in Liverpool in 1977 and 1978 were caused by PPNG. Thereafter the steady increase in PPNG infections to 5.6 per cent of all cases in 1982 was associated with marked changes in epidemiological pattern, plasmids and auxotypes.

Comparison of the substrate conformations in the active sites of papain, chymopapain, ficin and bromelain by resonance Raman spectroscopy.

The resonance Raman spectra of several enzyme-substrate intermediates of papain, chymopapain, ficin and bromelain are reported. The intermediates are dithioacyl enzymes formed during the catalyzed hydrolysis of N-acylglycine thionoester substrates. Interpretation of the resonance Raman spectra allows us to compare, for the first time, the substrate geometries in a series of functioning intermediates from different enzymes.