Mutational analysis of disulfide bonds in the trypsin-reactive subdomain of a Bowman-Birk-type inhibitor of trypsin and chymotrypsin--cooperative versus autonomous refolding of subdomains.

It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure. Accordingly, protein subdomains should behave as independent folding units. However, this prediction would underestimate the well-established structural significance of tertiary context and domain interfaces in proteins. The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis. The double-headed Bowman-Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study. The relative orientation of the two subdomains is stabilized by intramolecular and water-mediated hydrogen bonds and close ion pairs across a polar domain interface. The binary arrangement of a trypsin-reactive and a chymotrypsin-reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays. The functional consequences of five replacements in the S-S bond framework of the trypsin-reactive subdomain are analyzed in the present report. The mutants were subjected to refolding experiments in a refolding buffer and on trypsin-Sepharose as a template with complementary structure leading into a fully active state. The stability of the variants was assessed by means of subsequent equilibration experiments in solution. The mutants may be grouped into the following two classes: the class-I mutations located within beta-strand A are characterized by a breakdown of the trypsin- and the chymotrypsin-reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class-II mutations (beta-strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains. This points to a significance of polar interactions connecting the beta-strand A of the trypsin-reactive with the chymotrypsin-reactive subdomain. In conclusion, the polar domain interface appears as a major refolding unit of the Bowman-Birk inhibitor.

Ergocalciferol and cycloheximide in vivo stimulate protein kinase C of intestinal crypt cells.

Previous reports have suggested that 1,25-dihydroxychole-calciferol regulated cellular differentiation via its effects on protein kinase C activity. This study examined the in vivo effects of ergocalciferol on the activity of protein kinase C, and whether the differentiation of crypt intestinal cells is dependent on the activation of this enzyme. Ergocalciferol in saline was injected intramuscularly into rats and the animals sacrificed 24 hr after fasting. Protein kinase C specific activity was determined from the rate of incorporation of 32p-ATP into protamine. Injections of 60 micrograms ergocalciferol/200 g of body wt, raised protein kinase C specific activity to 59818 +/- 4010 (SEM, n = 5) cpm 32p-protamine/min/mg cell protein, compared with a control of 46173 +/- 4612 (P < 0.0005). Optimal specific activities were seen within 72 hr of injection. Administration of 120 micrograms ergocalciferol/200 g of body wt, raised the concentrations of serum calcium to 9.8 and 10.4 mg/dl following the intramuscular injection by 24 and 72 hr, respectively, compared with a control of 7.7 mg/dl. Actinomycin D (intramuscular, 100 micrograms/200 g of body wt) together with ergocalciferol (120 micrograms/200 g of body wt) reduced protein kinase C activity by 51% 24 hr after injection. Cycloheximide blocked the activation, but when injected alone stimulated endogenous protein kinase C activity by 34% 24 hr injection. The study shows activation of crypt protein kinase C by ergocalciferol. The inhibition of activation by actinomycin D and cycloheximide suggests the involvement of both transcriptional and translational processes in this activation.

Characterization of the functional role of E-box elements for the transcriptional activity of rat acetylcholine receptor epsilon-subunit and gamma-subunit gene promoters in primary muscle cell cultures.

The expression of gamma and epsilon subunits of the acetylcholine receptor from mammalian skeletal muscle is regulated independently during myogenic differentiation and innervation. Genomic DNA fragments containing 5'-flanking sequences of the epsilon-subunit and gamma-subunit genes were characterised by a series of 5' deletions fused to the chloramphenicol-acetyltransferase gene and transiently expressed by transfection of primary cultures of rat muscle cells and non-muscle cells. A 6.3-kb epsilon-subunit fragment can be reduced to yield a 270-bp fragment that confers 5-10-times higher expression levels in muscle cells compared to in non-muscle cells. The region composed of nucleotides -185 to -128 increases the transcriptional activity moderately while the 14-bp palindrome containing a single E box at nucleotides -88 to -83 may interact with the promoter but has no enhancer properties in muscle cells. From a 1.1-kb genomic fragment of the gamma-subunit gene, 167 bp were sufficient for muscle-specific expression. Two promoter-proximal E-box elements enhance promoter activity in muscle and mediate transactivation by myogenic factors. Myogenin and myf5 were much more efficient than MRF4 or MyoD1 which exerted only little transactivation. Cotransfection experiments show that increased expression of Id in primary muscle cells inhibits chloramphenicol-acetyltransferase expression mediated by the gamma-subunit gene promoter and support the view that myogenic factors play an important role in the transcriptional regulation of the gamma-subunit gene.

Two adjacent E box elements and a M-CAT box are involved in the muscle-specific regulation of the rat acetylcholine receptor beta subunit gene.

We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle-specific promoter element carrying a M-CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.

Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression.

Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma- and epsilon-subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5' flanking sequences of the gamma- and epsilon-subunit genes that confer muscle-specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma-subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle-specific transcriptional activity suggesting transactivation by helix-loop-helix proteins. The epsilon-subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non-muscle cells and thus may control the muscle-specific expression of the epsilon-subunit gene. The results suggest that CANNTG motifs together with their 3' and 5' flanking nucleotides provide binding sites for both activating as well as repressing trans-acting factors. These elements could thus contribute to the muscle-specific expression of AChR subunit genes.

Protein kinase C and leucine metabolism in intestinal crypt cells.

Intestinal cells were separated into fractions rich in villous or crypt cells. Alkaline phosphatase, present in villous cells, but absent from crypt cells, was used as a marker. Crypt cells were 3-6 times as active as villous cells in the metabolism of leucine or mevalonate. The previously reported stimulatory effect of albumin was twice as strong in crypt cells as that in villous cells. Reduced glutathione, spermidine HCl and ethanolamine (0.5-10 mM) did not replace albumin, the effect of which was maximal at 0.02 mM. Protein kinase C was shown to be present mainly in crypt cells.

Active transport system in human spermatozoa.

The presence of potassium influx into human spermatozoa was investigated through the use of radioactive K42 and Rb86. A gradient of potassium and sodium has been found between the spermatozoal midpiece-tail region and seminal plasma. The influx of potassium seemed to correlate with spermatozoal motility.

Induction of beta-galactosidase in Lactobacillus plantarum.

beta-galactosidase (beta-galactoside galactohydrolase, EC 3.2.1.23) is inducible in Lactobacillus plantarum by d-galactose or thiomethyl galactoside, and to a much lesser extent by lactose, isopropyl thiomethyl galactoside, and d-fucose. Isopropyl thiomethyl galactoside is a competitive inhibitor of the enzyme with a K(i) of 4.2 mM. The K(m) of the crude enzyme for o-nitrophenyl beta-d-galactoside is 0.87 mM. Induction also requires a source of energy and amino acids. Chloramphenicol and actinomycin D inhibited induction. d-Glucose, d-fructose and to a lesser extent maltose and d-mannitol inhibited enzyme synthesis. Methyl-alpha-d-glucopyranoside was not inhibitory. Glucose exerts its effect through its ability to exclude galactose or lactose entry into the cell. The uptake of lactose and the metabolism of galactose by preinduced cells is severely inhibited by glucose. But neither galactose nor lactose severely affected the uptake of glucose by preinduced cells. Thus, glucose acts through catabolite inhibition, i.e., transport of inducer rather than repression through transcription or related mechanisms. This is supported by the inability of cyclic nucleotides to relieve the inhibition produced by glucose or to stimulate induction. Furthermore, intracellularly produced glucose did not inhibit enzyme synthesis. Acetate and mevalonate, the precursors of membrane lipids, stimulate induction independently of their effect on growth. Homobiotin partially abolished the acetate effect but did not inhibit induction or growth.

Evidence against the presence of 3',5'-cyclic adenosine monophosphate and relevant enzymes in Lactobacillus plantarum.

Analysis of cells of Lactobacillus plantarum, starved or undergoing induction, showed no 3', 5'-cyclic adenosine monophosphate (cAMP). Neither adenyl cyclase nor 3', 5'-cAMP phosphodiesterase was detected in extracts. Extracts of L. plantarum did not inhibit these two enzymes of Escherichia coli K-12, strain W1435. Incubation of adenosine triphosphate (ATP)-U-(14)C with cells or various cell-free fractions of L. plantarum did not produce labeled 3', 5'-cAMP. Of various 3', 5'-cyclic and acyclic nucleotides tested, only 3', 5'-cAMP, ATP, and yeast adenylic acid stimulated l-arabinose isomerase. Yeast adenylic acid was two to four times as effective as 3', 5'-cAMP or ATP. 2', 3'-cAMP was not effective.

The biosynthesis of C 55 polyprenols by a cell-free preparation of Lactobacillus plantarum.

A cell-free supernatant of lysates of Lactobacillus plantarum catalyses the synthesis of lipids from [2-(14)C]mevalonate. Of the added mevalonate, 7.5% is incorporated into lipids, which were fractionated into five components. About 4% of the radioactivity in these lipids co-chromatographs with compounds shown by mass spectrometry, n.m.r. and i.r. spectroscopy to be C(55) polyprenols, and about 2% co-chromatographs with a hexamer. The rest of the radioactivity is in more complex fractions. Analysis by mass spectrometry, n.m.r. and i.r. spectroscopy shows that the major C(55) polyprenol is undecaprenol, accompanied by an isomer containing one reduced isoprene unit. A Kuhn-Roth degradation of [(14)C]polyprenols indicates that the supernatant catalyses synthesis of these compounds de novo.

Mevalonate metabolism in supernatant fractions of lysates of Lactobacillus plantarum.

The supernatant fractions of lysates of Lactobacillus plantarum metabolize mevalonate into lipids. Adenosine triphosphate and uridine, as well as related compounds, and reduced nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide stimulate this process. To obtain very active supernatant fractions, the method of lysis is modified to include polyamines during lysozyme treatment of cells and subsequent shocking with citrate buffer.

Osmotic fragility of lysozyme- and thiol-treated Lactobacillus plantarum.

Reduced but not oxidized thiols increased the sensitivity of lysozyme-treated cells of Lactobacillus plantarum to lysis by osmotic shock.

The biosynthesis of squalene and sterols by the adipose tissue of rat, sheep and man.

1. The subcutaneous and omental adipose tissue of man, the epididymal fat pads of the rat and the fat tail of the Syrian sheep incorporate mevalonic acid into non-saponifiable lipids. 2. Time studies showed that the rates of decarboxylation of mevalonic acid and synthesis of non-saponifiable lipids slightly decline after 20min. but subsequently remain linear for 6hr. 3. About one-half of the incorporated radioactivity in the non-saponifiable lipids was in squalene, 20% in lanosterol and cholesterol, and the remainder in unidentified substances.