Influence of cyclic nucleotides on the processing of the carbohydrate part of the alpha-subunit of human choriogonadotropin by first trimester human placenta tissue

In pulse-chase experiments ([35S]Met as radioactive label) 4 intracellular forms of the alpha-subunit (apparent molecular weights of 11, 16.5, 19.5, and 23.4 kDa) were observed whereas almost no label was incorporated into the beta-subunit. The 23.4 kDa form was secreted as free alpha-subunit, the others were precursors of the alpha-subunit contained in secreted human choriogonadotropin. The rate-limiting step seemed to be the processing of the 19.5 kDa precursor by alpha-mannosidase II. 8-bromo-cAMP increased the total amount of intracellular forms of the alpha-subunit and accelerated significantly the velocity of all glycosylation steps. It seemed to cause a higher efficacy of the alpha-mannosidase II reaction. In the presence of 8-bromo-cAMP intracellular as well as extracellular alpha-subunits showed a higher sialic acid content.     

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.     

Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures

We have reported a rapid method for the quantitation of proteins secreted in culture media ([12.]). Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture. The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin. Since heparin is known to affect certain cellular functions, the fates of [³⁵S]methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments. Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin. At 8 h during the chase, there was a 40–50% reduction in fibrinogen-antigen in spent culture medium lacking heparin. The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis. In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen. The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation. Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.     

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.     

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.     

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.     

Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion

To identify the rate-limiting step(s) in the hepatic production of very low density lipoproteins (VLDL), we investigated the intracellular distribution and rate of intracellular transport of de novo synthesized apolipoprotein B (apoB). For all secretory proteins examined (i.e. albumin, large molecular weight apoB, and small molecular weight apoB) the rough and smooth microsomes contained the majority of intracellular de novo synthesized protein, while the Golgi subfraction contained 10% or less. Pulse-chase analysis of the intracellular movement of apoB and albumin showed that the first order rate constant (in terms of half-life) describing the rate of movement out of the smooth and rough microsomes determined the overall rate of movement out of the cell. These data suggest that movement out of the endoplasmic reticulum, the site where VLDL is assembled, determines the overall rate of secretion. Furthermore, compared to albumin, the rate of intracellular transport of apoB was approximately two times slower suggesting that processing unique to VLDL apoB occurring in the endoplasmic reticulum was responsible. Additional studies show that essentially all of the de novo synthesized 35S-labeled albumin (produced from a pulse of [35S]methionine) lost from the cell during the chase period could be recovered in the culture medium. In contrast, much less of large molecular weight apoB (36%) and small molecular weight apoB (60%) was recovered in the culture medium. Since these cultured rat hepatocytes do not take up or degrade newly secreted apoB, these data suggest that a significant amount of apoB is degraded intracellularly.     

Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes

Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorporation of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBH with an intra-cellular retention half-time of 50 min (initial half-life of decay, t1/2 = 25 min) compared with 85 min in control (t1/2 = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin-treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively. When hepatocyte monolayers were continuously labeled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBH was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase. Insulin inhibited the secretion of immunoassayable apoB but not labeled apoB. Results demonstrate that 1) insulin inhibits synthesis of apoB from [35S]methionine, 2) insulin stimulates degradation of freshly translated apoB favoring apoBH over apoBL, and 3) an intracellular pool of apoB, primarily apoBL, exists that is largely unaffected by insulin. Overall, insulin action in primary hepatocyte cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.     

Intracellular immature subunits of human chorionic gonadotropin in first trimester placental cells: purification and characterization

As we previously reported [Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i.e., the 21 kDa form of alpha-subunit and the 23 and 19 kDa forms of beta-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracellular hCG consisting of these subunits, based on gel filtration, was approximately 200 kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of alpha-subunit as well as the 23 and 19 kDa forms of beta-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and alpha-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not O-linked sugar chains of the type present in mature beta-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary beta-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of beta-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of the steady-state RNA levels among the alpha-subunit of prolyl 4-hydroxylase and the alpha 1 and alpha 2 chains of type I collagen during growth of chicken embryo tendon fibroblasts.

The relative steady-state levels of RNAs encoding type I collagen and prolyl 4-hydroxylase were examined in exponentially growing primary cultures of chicken embryo tendon fibroblasts. The RNA levels of the alpha 1 and alpha 2 chains of type I collagen were maximal when the fibroblasts reached the confluent state. The RNA levels of the alpha-subunit of prolyl 4-hydroxylase were also maximal at confluency and rose and fell with the RNA levels of the two collagen chains. The RNA levels of the beta-subunit of prolyl 4-hydroxylase did not correlate with the changes observed for the alpha-subunit or for either chain of type I collagen. The RNA levels of the beta-subunit were slightly higher than the RNA levels of the alpha-subunit. These results support our hypothesis that the synthesis of the alpha-subunit and thus the association of newly synthesized alpha-subunits with pre-existing beta-subunits is the rate-limiting factor in determining prolyl 4-hydroxylase activity in cultured cells.     

Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges.

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.     

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.     

Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme

A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg). The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit. We did not observe any proteolytic cleavage in the course of the heat solubilization. The enzyme is competively inhibited by azide, but not by chlorate. It exhibits a K(M) value of 3.2 mM for nitrate. We compare the enzymatic and electron paramagnetic resonance (EPR) spectroscopic properties of the alphabeta form with the alphabetagamma holoenzyme which resides in the membrane and can be prepared by detergent extraction. The nearly identical EPR spectra for the Mo(V) signal of both enzyme preparations show that the active site is unaffected by the heat step. The factors influencing the binding of the alpha- and beta-subunit to the gamma-subunit are discussed.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.     

Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).     

Subunit structure of high molecular weight mouse nerve growth factor

Studies from several laboratories have shown that mouse submandibular glands and mouse saliva contain nerve growth factor (NGF) as part of a high molecular weight oligomeric macromolecule composed of three different subunits, termed alpha, beta, and gamma. The beta-subunit is the nerve growth-promoting protein. The gamma-subunit is a serine protease class enzyme of highly restricted substrate specificity. The alpha-subunit has no known function. This high molecular weight form of nerve growth factor is also a Zn(II)-containing metalloprotein. In the present study, measurements of multiple physicochemical parameters have been used to deduce the subunit structure of high molecular weight NGF. Results demonstrate that it contains two alpha-, one beta- and one gamma-subunit together with one tightly bound Zn(II) ion per molecule.     

Human chorionic gonadotropin alpha-subunit of normal placenta: characterization of synthesis and association with beta-subunit

The forms of human chorionic gonadotropin (hCG) alpha-subunit synthesized and released by normal placental tissue were examined in explants of first trimester placenta that had been incubated for 30 min with [35S]methionine and then incubated for 6 h in medium containing unlabeled methionine. The media and tissue extracts collected at 0, 0.5, 1, 2, 4, and 6 h after the exposure to [35S]methionine were chromatographed on Sephadex G-100 and the amounts of radioimmunoassayable alpha-subunit and immunoprecipitable 35S-labeled alpha-subunit were determined. In tissue extracts, a single form of alpha-subunit was observed at 0 h that had an apparent molecular weight smaller (Ve/Vo = 1.90) than that of a urinary hCG alpha reference preparation (Ve/Vo = 1.81). With chase times of 0.5, 1, 2, and 4 h, a second peak of alpha-subunit was detected that had a larger apparent molecular weight (Ve/Vo = 1.68), and the ratio of large to small forms increased progressively with incubation time. In contrast to that in the extracts, the 35S-labeled alpha-subunit in the culture medium consisted entirely of the large form. Large and small intracellular forms of free alpha-subunit exhibited less than 2% recombination with beta-subunit, as evidenced by gonadotropin receptor binding activity. These studies suggest that normal placental tissue synthesizes a small precursor form of free hCG alpha-subunit that is converted to a larger form prior to secretion and that the free forms of alpha-subunit do not bind to purified hCG beta-subunit.     

Sequencing, expression, and enzymatic characterization of beta-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells

The lysosomal enzyme, beta-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an alpha beta-subunit heterodimer and HexB a beta-subunit homodimer. Both isoforms can remove nonreducing beta-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as G(M2) gangliosides as well. beta-Hexosaminidase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary culture on stimulation with secretagogs. To further characterize the enzyme, the alpha- and beta-subunit mRNA expression was explored in rabbit lacrimal gland tissue as well as in cultured cells. Possible correlation between mRNA expression and HexA specific enzymatic activity was also investigated. Because existing beta-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the alpha- and beta-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the beta-subunit, showing an even mRNA expression between tissue and cultured cells, the level of alpha-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total beta-hexosaminidase as well as HexA activity was observed in cultured cells compared to tissue. Enzymatic activity assays also revealed that HexA is the dominating isoform of beta-hexosaminidase in lacrimal gland and cultured acinar cells.     

Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967). Pulse-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments.