Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Antithrombin III mRNA in adult rat liver and kidney and in rat liver during development

Using Northern blots the size of antithrombin III (AT III) mRNA in rat liver was found to be 1650 nucleotides. Adult rat kidney also contained a slightly smaller mRNA at about 20% the level in liver. The ontogeny of AT III mRNA in the liver was assessed by dot blot hybridization. The mRNA was detectable at the earliest age examined (14th day of gestation) at about 15% of the adult levels. After the 17th day of gestation the levels of antithrombin III mRNA rise reaching 50% of adult levels at birth. After birth the mRNA levels rise to 75% of adult levels by the 5th day and reach adult levels by 40 days after birth. We suggest that foetal AT III is produced by both the foetal liver and by placental transfer of the maternal inhibitor.     

A rapid method for the purification of D-amino acid oxidase of Trigonopsis variabilis by hydrophobic chromatography

Summary A relatively simple method has been described for the rapid purification of D-amino acid oxidase from Trigonopsis variabilis by hydrophobic chromatography on Phenyl-Sepharose CL-4B and negative adsorption on DEAE-cellulose. The purified enzyme had a specific activity of 22–24 units at 25°C and exhibited three bands on enzymatic staining.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.     

Correction of the published sequence for the human proteolipid protein gene

Summary In the human proteolipid protein gene, the base sequence of the intronic region 5 to exon 6 was found to be 5-ctctttcattttcctgcag-3 and not 5-ctctttt-cattttcctgcag-3 as previously reported.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Immobilization of fumarase by entrapment of rat liver mitochondria in polyacrylamide gel using gamma rays

A stable immobilized preparation of fumarase (EC 4.2.1.2) was obtained by entrapment of rat liver mitochondria in acrylamide polymerized by using gamma irradiation (100 kR). The enhanced stability and the efficiency of the entrapped enzyme have shown potential for repeated use for the production of L-malic acid from fumaric acid. The possible formation of succinic acid in the system could be controlled by incorporating malonate along with detergents such as sodium deoxycholate or sodium dodecylsulfate in the reactor system.