Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties.

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.     

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein.     

Experimental approaches to the study of the biogenesis of mammalian mitochondrial proteins

Mitochondrial proteins are synthesized in mitochondria and on cytosolic ribosomes. Several approaches used to establish the site of synthesis and the identity of mitochondrially synthesized proteins are described. These include the specific inhibition of mitochondrial translation by inhibitors or mutation and the specific elimination of cytosolic translation either by using isolated mitochondria or specific inhibitors. Experimental approaches to study the import of proteins into mitochondria are also discussed.     

Preliminary crystallographic data for plastocyanins from an alga (Enteromorpha prolifera) and from cucumber (Cucumis sativus)

The plastocyanins from a green alga (Enteromorpha prolifera) and cucumber (Cucumis sativus) have been crystallized. Crystal data are as follows: E. prolifera plastocyanin, space group I4, a = b = 53.9 A, c = 59.4 A, Z = 8; C. sativus plastocyanin, space group P4(1) (or P4(3) ), a = b = 66.7 A, c = 46.0 A, Z = 8. Accordingly, the asymmetric units of the crystals contain one and two molecules, respectively.     

Turnover rates of amino acid neurotransmitters in regions of rat cerebellum

The turnover rates of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, alanine, serine, and glycine were measured in five regions of rat cerebellum. Turnover rates of the putative neurotransmitters (aspartate, glutamate, and GABA) were 2-20-fold higher than those of alanine and serine, and generally consistent with the proposed neurotransmitter functions for these amino acids. However, glutamate turnover was high and similar in magnitude in the deep nuclei and granule layer, suggesting possible release, not only from parallel fibers, but from mossy fibers as well. The differential distribution of turnover rates for GABA supports its neuronal release by Purkinje, stellate, basket, and Golgi cells, whereas aspartate may be released by both climbing and mossy fibers. The distribution of glycine turnover rates is consistent with release from Golgi cells, whereas alanine may be released from granule cell parallel fibers. Turnover rates measured in two other motor areas, the striatum and motor cortex, indicated that utilization of these amino acid neurotransmitters is differentially distributed in brain motor regions. The data indicate that turnover rate measurements may be useful in identifying neurotransmitter function where content measurements alone are insufficient.     

Additional evidence for fragile X activity in heterozygous carriers.

The result of a previous study showing an association between mental development and fragile X activity in heterozygous females is given further support by similar investigations of three additional kindreds. The increased frequency of demonstrable fragile X chromosomes in mentally retarded females appears to be due to an increase in the active fragile X while the inactive marker X remains at a similar low frequency in all heterozygotes whether retarded or not. The frequencies of the active fragile X separated the normal and abnormal subjects into two distinct populations. The suggested inverse correlation between the number of lymphocytes with detectable fragile X chromosomes and advancing age can be attributed to ascertainment biases.     

Twinning rate in spontaneous abortions.

An unselected series of spontaneous abortions and their mothers were karyotyped with Q-bands to obtain a frequency of twin conceptions lost during the first trimester. Among 661 spontaneous abortions, 15 twin pairs were identified including two sets of conjoined twins. Analysis of Q-band variants permitted the exclusion of cases with two cell lines that could be attributed to maternal contamination or mosaicism. The twinning rate among spontaneous abortions was 1/44 compared with 1/103 live births and stillbirths in the Ontario population. If Weinberg's differential method is applied to these data, the frequency would be as high as 1/30 under the assumption that the incidence of monozygotic twins among abortions is the same as that for live births.     

B-chromosome behaviour in Phaulacridium vittatum

Nine individuals of Phaulacridium vittatum in a single population sample of 1250 males collected at Crookwell, N. S.W. contained a single supernumerary chromosome. One further individual had two B-chromosomes. The supernumerary in question was larger than any of the standard set and was distinguishable from them at all stages of mitosis and meiosis. Like the X-univalent the B-chromosome was heteropycnotic at the onset of meiosis but differed from it in size, structure and behaviour. During first prophase single supernumeraries were frequently associated with the X in a non-homologous fashion as a consequence of their precocity. All such associations lapsed before first metaphase and the X and the B moved at random with respect to one another at first anaphase. In the one individual with two supernumeraries the two B-chromosomes showed regular pairing and segregated in a conventional manner. In all these respects the Crookwell supernumerary differed markedly from a morphologically identical B-chromosome present in a population of the same species from Hobart, Tasmania studied by Jackson and Cheung in 1967. Whether these differences in the behaviour of the B-chromosome in the two populations determines the higher frequency of the supernumerary in Hobart (11.3%) as compared with Crookwell (0.8%) remains to be resolved.     

Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon

Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed -CO(2)... NH(2) (-) pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp'-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.