Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

1,6-Diamino-2,5-anhydro-1,6-dideoxy-dl-glucitol and some derivatives thereof

1,6-Diamino-2,5-anhydro-1,6-dideoxy-dl-glucitol dihydrochloride and some derivatives were synthesized from 3,5-di-O-acetyl-1,6-dibromo-1,6-dideoxy-d-mannitol. Introduction of the 2,5-anhydro ring and subsequent replacement of the terminal bromine atoms by azide gave low yields of the diazide; therefore, a reverse reaction-sequence was applied. The azido groups were reduced with hydrogen sulfide-pyridine, and the amino groups formed were methylated by using formaldehyde-formic acid and subsequently treating with borohydride. According to ¹³C-n.m.r. investigations, the symmetrically substituted, 2,5-anhydroglucitol derivatives are present mainly in the ⁴T₃ [“north” (N) type of twist] conformation, whereas the analogous l-iditol derivatives mainly adopt the ³T₄ [“south” (S)] type. The different quaternary salts obtained on methylation of the corresponding 1,6-bis(dimethylamino)derivatives with methyl iodide (aiming at the structure of epi-muscarine) showed no muscarine-like, biological activity.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Kinetic studies of the applicability of the common site concept to the 3β-hydroxysteroid dehydrogenase: 5-ene-3-ketosteroid isomerase activities of human placental microsomes

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with K_M values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with K_I values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with K_I values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with K_I values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the K_M for pregnenolone as a 3β-HSD substrate, had no effect on the K_M for the isomerase substrate 5-pregnene-3, 20-dione.It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.